Expression, intracellular localization, and mutation of EGFR in conjunctival squamous cell carcinoma and the association with prognosis and treatment

Atsushi Sakai; Mizuki Tagami; Anna Kakehashi; Atsuko Katsuyama-Yoshikawa; Norihiko Misawa; Hideki Wanibuchi; Atsushi Azumi; Shigeru Honda

doi:10.1371/journal.pone.0238120

. 2020 Aug 24;15(8):e0238120. doi: 10.1371/journal.pone.0238120

Expression, intracellular localization, and mutation of EGFR in conjunctival squamous cell carcinoma and the association with prognosis and treatment

Atsushi Sakai ¹, Mizuki Tagami ^1,^2,^*, Anna Kakehashi ³, Atsuko Katsuyama-Yoshikawa ^2,⁴, Norihiko Misawa ¹, Hideki Wanibuchi ³, Atsushi Azumi ², Shigeru Honda ¹

Editor: Sanjoy Bhattacharya⁵

PMCID: PMC7444806 PMID: 32833992

Abstract

Purpose

Conjunctival squamous cell carcinoma (SCC) is primarily treated with surgical resection. SCC has various stages, and local recurrence is common. The purpose of this study was to determine molecular localization of epidermal growth factor receptor (EGFR) and the possibility of EGFR as a biomarker for the management of conjunctival SCC.

Methods

In this retrospective study, we performed immunohistochemistry to evaluate EGFR expression and localization in tumor cells, EGFR mutation-specific expression (E746-A750del and L858R), and human papillomavirus expression in a series of 29 conjunctival SCCs.

Results

All 29 tumors in our cohort were EGFR positive (100%). Twenty-one of 29 tumors (72%) showed focal EGFR staining, and seven (28%) showed diffuse EGFR staining. In addition, we calculated the percentages of the two most important mutations in EGFR (exon 19 746-A750del (8/29, 27.5%), exon 21 (L858R mutant (2/29, 6.8%)) in conjunctival SCCs. We observed that the translocation of EGFR from the membrane into the cytoplasm was related to clinical prognosis, as we detected correlations between EGFR cytoplasmic staining and final orbital exenteration and between decreased EGFR membrane staining and progression-free survival.

Conclusions

EGFR is important in the pathology of ocular surface squamous neoplasia including SCC and is a prognostic factor. Increased understanding of EGFR mutations may have important implications for future treatment options.

Introduction

Ocular surface squamous neoplasia (OSSN) includes several diseases such as conjunctival premalignant dysplasia, carcinoma in situ, and invasive conjunctival squamous cell carcinoma (SCC) [1]. The annual incidence of OSSN was 0.53 cases/million/year (conjunctival intraepithelial neoplasia: 0.43 cases/million/year; SCC: 0.08 cases/million/year) in the United Kingdom [2, 3]. In the United States, the rate of SCC is 5-fold higher among males and whites [4].

Other previous research revealed that the risk increases with exposure to direct daylight and in outdoor workers. Meta-analysis demonstrated an association with human immunodeficiency virus (odds ratio, 6.2) and human papillomavirus (HPV) (odds ratio, 2.6) [4]. However, no large epidemiological studies have been performed on people living in the Far East.

Scholz et al. examined clinicopathological factors and biomarkers and identified promoter mutations in telomerase reverse transcriptase in 44% of 48 samples of conjunctival OSSN associated with ultraviolet light induction [5]. Recent research demonstrated that PD-L1 is expressed in almost half of conjunctival SCC cases and noted the potential application of immune checkpoint blockade as a treatment strategy for conjunctival SCC [6].

Molecular targeted therapy is now used to treat most carcinomas, and its use is continuing to increase [7]. Uveal melanoma also has recently been reported in the ocular oncology area [8]. Gefitinib is a relatively old tyrosine kinase inhibiter (TKI) that is used as a molecular targeted therapy, and its effects have been reported in various carcinomas. On the other hand, no basic clinical studies on ocular tumors have been reported [9–11]. In our current study, we investigated epidermal growth factor receptor (EGFR) expression in our cases to assess the possible effect of gefitinib. We also examined the molecular expression and intracellular localization of EGFR in conjunctival SCC in East Asian patients.

Materials and methods

Selection of cases and collation of clinicopathologic data

This study was approved by the Institutional Review Boards of Osaka City University and Kobe Kaisei Hospital and adhered to the tenets of the 1964 Declaration of Helsinki. Written informed consent was obtained from all patients before enrollment. We identified 29 patients treated by ophthalmologists (AA, MT) between November 2007 and July 2018 from whom we were able to procure tissue blocks with residual tumor. For each patient, we collected demographic features (age at initial diagnosis and at presentation to our institution, and sex) and primary tumor features (disease status at presentation [primary or recurrent] and in situ versus invasive disease). The American Joint Committee on Cancer (AJCC) stage, local recurrence (anatomic site and date), metastases (regional or distant and date), vital status at last follow-up, cause of death, types of surgery, and adjuvant therapy were also recorded.

Immunohistochemistry (IHC)

Immunohistochemical studies for EGFR and HPV were performed on 6-μm-thick tissue sections using the following antibodies: anti-human EGFR rabbit monoclonal antibody (clone: SP84; #414R-14; CELL MARQUE, Rocklin, CA, USA), anti-HPV mouse monoclonal antibody (clone: K1H8; ab75574; abcam, Cambridge, UK), horseradish peroxidase-conjugated anti-rabbit IgG (H+L) goat polyclonal antibody (HISTOFINE #424134, Nichirei Corporation, Tokyo, Japan), and horseradish peroxidase-conjugated anti-mouse IgG (H+L) goat polyclonal antibody (HISTOFINE #424144, Nichirei Corporation).

EGFR mutation-specific immunohistochemical staining was performed on 29 cases. As primary antibodies, we used EGFR E746-A750del (#2085, Cell Signaling Technologies, Danvers, MA, USA) and EGFR L858R (#3197, Cell Signaling Technologies), which were manually applied to the slides. Stained sections were viewed with an Olympus BX53+DP74.

As controls for staining, benign conjunctival lesions were also stained for EGFR, and colon cancer samples were stained as a positive control.

Image analysis

Slides immunostained for EGFR, EGFR mutations, and HPV were evaluated in a blinded manner by two specialists (MT and AK). EGFR expression was visually estimated as the percentage of tumor cells with complete or partial membranous staining. Tumors with EGFR staining in ≥50% of tumor cells were considered the diffuse staining type (diffuse type), and those with <50% of tumor cells were considered the focal staining type (focal type). The presence or absence and intensity of cell membrane staining were semi-quantitatively divided into groups with a score of 0–3 (0: none, 1: weak, 2: strong, 3: very strong). The presence or absence and intensity of cell cytoplasmic staining were also divided into groups with a score of 0–3 and semi-quantitatively analyzed (0: none, 1: weak, 2: strong, 3: very strong). EGFR mutation-specific immunostaining was divided into two groups: those with immunostaining that was clearly present and those without immunostaining.

Slides immunostained for HPV were assessed with visual evaluation for the presence of punctate nuclear signals within tumor nuclei at 400× magnification and were scored as positive or negative.

EGFR expression in tumors

EGFR expression in the tumor was analyzed with NanoString analysis. Archival formalin-fixed paraffin-embedded tumor tissue was retrieved and manually macrodissected. Total mRNA was isolated from the macrodissected tumor tissues using a Qiagen miRNeasy kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. The RNA sample was quantified with NanoDrop (Thermo Scientific, Wilmington, DE, USA) and regarded as adequate if it contained 400 ng at minimum. The sample was subsequently analyzed with the nCounter PanCancer Progression Panel (NanoString, Seattle, WA, USA) according to the manufacturer’s instructions [12]. NanoString data processing was done with the R statistical programming environment (v3.4.2). Considering the counts obtained for positive control probe sets, raw NanoString counts for each gene were subjected to technical factorial normalization, which was carried out by subtracting the mean counts plus two times the standard deviation from the CodeSet inherent negative controls. Subsequently, biological normalization using the included mRNA reference genes was performed. Additionally, all counts with P > 0.05 after a one-sided t-test versus negative controls plus two times the standard deviation were interpreted as not expressed over basal noise.

Statistical analysis

The clinical and histopathologic characteristics were summarized using descriptive statistics. Correlations between immunohistochemical, demographic, and clinicopathologic factors were assessed using the Wilcoxon rank sum and Fisher's exact tests. Progression-free survival (PFS) was defined as the time from surgery to disease recurrence or death from any cause. Cox regression modeling was used to evaluate correlations between clinicopathologic and immunohistochemical features and survival outcomes. Statistical analyses were performed using SPSS Statistics version 22 software (IBM Japan, Tokyo, Japan). Values of P < 0.05 were considered statistically significant.

Results

Clinicopathologic findings of our cohort are summarized in Table 1. All 29 patients in our cohort (100%) were East Asian, and included 15 men and 14 women with a mean age at presentation of 77.4 years. Fourteen patients (48%) had invasive SCC, and 15 (52%) had an in situ tumor. Primary orbital exenteration was necessary for local disease control in two patients (6%), and two patients (6%) underwent additional orbital exenteration. Nine patients (31%) underwent adjuvant therapy, most commonly additional local surgery. Topical chemotherapy and radiation therapy were performed in one patient in the adjuvant therapy group. Of this group, one patient died with disease 11 months after diagnosis of regional and lung metastases; the other patient was alive without disease at 44 months after diagnosis of regional metastases. Two patients (6%) died, one of which was due to conjunctival SCC (described above). Nine patients (31%) experienced local recurrence after curative surgery.

Table 1. Clinicopathologic findings of 29 cases of conjunctival squamous cell carcinoma.

	All (n = 29) n (%)
Age, years; mean (range)	77.4 (63–98)
Sex
Male	15 (52)
Female	14 (48)
Follow-up after primary surgery; months (range)	40.9 (3–135)
T-stage (AJCC)
Tis	15 (52)
T1	3 (10)
T2	3 (10)
T3	7 (25)
T4	1 (3)
Primary surgery type
Local excision	27
Orbital exenteration	2
Adjuvant therapy
No	20 (69)
Yes	9 (31)
Additional excision	7
Topical chemotherapy	1
Radiation therapy	1
Immunohistochemical markers
HPV status in tumor cells
Negative	22 (76)
Positive	7 (24)
EGFR expression in tumors
Diffuse staining	8 (27)
Focal staining	21 (73)
Negative	0 (0)
Cell membrane EGFR expression in tumors
Very strong	1 (3)
Strong	21 (72)
Weak	7 (25)
Negative	0 (0)
Cell cytoplasm EGFR expression in tumors
Very strong	4 (14)
Strong	6 (20)
Weak	19 (66)
Negative	0 (0)
Outcome
Orbital exenteration
Yes	4 (14)
No	25 (86)
Local recurrence after curative therapy
Yes	9 (31)
No	18 (69)
Metastasis
Distant	0 (0)
Regional + distant	1 (3)
Regional	1 (3)
None	27 (94)
Vital status at last follow-up
Dead	2 (6)
Alive	27 (94)
Cause of death
Conjunctival SCC (metastasis)	1 (50)
Other	1 (50)

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All 29 tumors were EGFR positive (100%) in our cohort. Twenty-one of 29 tumors (72%) showed focal EGFR staining, and seven (28%) showed diffuse EGFR staining (Fig 1). Analysis of EGFR intracellular staining patterns showed scores of 1.72 for membrane staining and 1.48 for cytoplasmic staining. No significant difference was found between carcinoma in situ (Tis) and invasive carcinoma (Tadv) (Table 2). No significant difference was found in the score depending on the stage. EGFR expression in colon cancer was used as a positive control (Fig 2A).

Fig 1 — Focal EGFR staining (A) and diffuse EGFR staining (B) (Scale bar: 50 μm). Inset: corresponding field in a hematoxylin-eosin-stained section. Membrane staining (very strong: 3) (C) and cytoplasm staining (very strong: 3) (D) (Scale bar: 20 μm).

Table 2. Staining patterns of EGFR.

		Staining patterns (N = 29)
Cell membrane		0	1	2	3	Average	P
Tis (in situ)	N = 15	0	4	11	0	1.73	0.93
Tadv (invasive)	N = 14	1	3	9	1	1.71
					total	1.72
Benign tumor	N = 7	1	4	2	0	1.28	0.30
Cell cytoplasm		0	1	2	3	Average	P
Tis (in situ)	N = 15	0	9	5	1	1.46	0.90
Tadv (invasive)	N = 14	0	10	1	3	1.5
					total	1.48
Benign tumor	N = 7	6	1	0	0	0.14	<0.01^*

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*p value based on the non-paired t-test.

Fig 2 — (A) EGFR expression in colon cancer as a positive control (Scale bar: 50 μm). (B) EGFR expression in a control benign lesion, pinguecula (Scale bar: 50 μm). Inset: corresponding field in a hematoxylin-eosin-stained section.

On the other hand, seven benign conjunctival lesions (three pinguecula, three pterygium, one dermoid cyst) showed partial weak positive staining in conjunctival squamous epithelial cells, especially on the cell membrane (Fig 2B). In addition, cytoplasmic staining was seen in only one case. Benign cases showed scores of 1.28 for membrane staining and 0.14 for cytoplasmic staining. Cytoplasmic staining patterns were significantly different in benign compared to SCC cases (P < 0.01) (Table 2). The correlation between EGFR staining (focal or diffuse) and EGFR localization (cytoplasmic staining group) was not significantly different, but the diffuse EGFR group tended to have a higher score (p = 0.38 and 0.12, respectively) (Table 3).

Table 3. EGFR staining and localization.

	EGFR Focal(N = 21)	EGFR Diffuse(N = 8)	P
Cell Membrane	1.8±0.9	1.5±0.9	0.38
Cell cytoplasmic	1.3±0.6	1.8±0.8	0.12

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EGFR E746-A750 del and EGFR L858R expression were assessed with immunohistochemistry in all 29 patients (Fig 3). The mutation at exon 19, EGFR E7446-A750 del, was confirmed in 8/29 (27.5%) cases, and that at exon 21, EGFR L858R point mutation, was confirmed in 2/29 (6.8%) cases with IHC (Table 4). The relationship between EGFR mutation and EGFR staining (focal or diffuse) was determined using univariate linear regression analysis with correction for age (P = 0.559).

Table 4. Summary of EGFR E746-A750 del and EGFR L858R point mutations.

Mutation	N = 29 (%)	Age (y)	Sex (M/F)	T stage	EGFR staining patterns (diffuse/focal)	EGFR localization score (membrane/cytoplasmic)
Exon 19 EGFR E746-A750 del (N = 8)	8/29 (27.5%)	75.8	3/5	T3: 4	2/6	1.6/1.5
				T2: 2
				Tis: 2
Exon 21 EGFR L858R point mutation (N = 3)	2/29 (6.8%)	73.0	1/1	T3: 1	1/2	1/3
Exon 21 EGFR L858R point mutation (N = 3)	2/29 (6.8%)	73.0	1/1	Tis: 1	1/2	1/3

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M: Male, F: Female.

Regarding EGFR expression in tumors, we compared the Tis and Tadv groups according to AJCC T grading (n-4, 4). No significant difference was found (P = 0.162) (Fig 4).

The majority of patients in our cohort were HPV negative (n = 22; 75%) (Table 1). The positive rate of HPV immunoreactivity increased with increases in AJCC T grading, but the correlation was not statistically significant.

The Cox regression model was used to examine and analyze the relationship between long-term prognosis including orbital exenteration and PFS and the clinicopathological status, EGFR staining pattern, and EGFR mutation. Univariate Cox regression analyses revealed significant correlations between EGFR cytoplasmic staining and final orbital exenteration (hazard ratio (HR): 4.2, P = 0.036) (Table 5). Additionally, a significant correlation was seen between the T stage (AJCC) and PFS, and between EGFR membrane staining and PFS (HR: 13.1, 0.23, P = 0.025, P = 0.015, respectively) (Table 6). Local recurrence, distant metastasis rate, and overall survival rate were not statistically significant. In addition, the EGFR mutation was not significantly correlated with final orbital exenteration or PFS (Tables 5 and 6).

Table 5. Relationship between orbital exenteration and clinicopathologic and molecular factors.

Univariate analysis
Variables	N = 29	HR	95% CI	P
Age	Mean 77.3 years	1.68	0.834–3.406	0.146
Sex	Male 15, Female 14	0.925	0.129–6.605	0.938
T-stage (AJCC)	Tis, T1, T2: 21/T3 ≥8	7.551	0.785–72.650	0.080
EGFR staining	Focal 21/Diffuse 8	7.21 × 10²	0.001–82.9 × 10	0.365
EGFR membrane staining	Very strong 1/Strong 21/Weak 7/Negative 0	0.415	0.121–1.429	0.164
EGFR cytoplasmic staining	Very strong 4/Strong 6/Weak 19/Negative 0	4.206	10.97–16.122	0.036^*
*EGFR* mutation	Exon 19 E746-A750 del 8/Exon 21 L858R point mutation 2	0.582	0.096–3.545	0.558
HPV positive	Positive 7/Negative 22	0.032	0.00–5.07 × 10²	0.485

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CI indicates confidence interval; HR hazard rate.

Statistically significant differences are underlined.

*p value based on the Cox proportional hazard model.

Table 6. Relationship between PFS and clinicopathologic and molecular factors.

Univariate analysis
Variables	N = 29	HR	95% CI	P
Age	Mean, 77.3 years	1.15	0.970–1.384	0.104
Sex	Male 15/Female 14	0.611	0.101–3.690	0.592
T-stage (AJCC)	Tis, T1, T2 21/T3 ≥8	13.11	1.384–1.24 × 10²	0.025^*
EGFR staining	Focal 21/Diffuse 8	3.635	0.685–19.289	0.130
EGFR membrane staining	Very strong 1/Strong 21/Weak 7/Negative 0	0.237	0.074–0.759	0.015^*
EGFR cytoplasmic staining	Very strong 4/Strong 6/Weak 19/Negative 0	2.813	0.993–7.973	0.052
*EGFR* mutation	Exon 19 E746-A750del 8/Exon 21 L858R point mutation 2	33.512	0.00–1.91 × 10⁷	0.604
HPV positive	Positive 7/Negative 22	0.459	0.052–4.077	0.484

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CI indicates confidence interval; HR hazard rate.

Statistically significant differences are underlined.

*p value based on the Cox proportional hazard model.

Discussion

To the best of our knowledge, this is one of the first studies to survey the prevalence of EGFR mutations and intracellular localization in conjunctival SCC and to evaluate the prognostic significance of tumor cells that express EGFR in conjunctival SCC.

In this study, we found that the tumor tissue of all conjunctival SCCs (100%) expressed EGFR. In addition, we determined the percentages of the two most important mutations in EGFR (exon 19 746-A750del (8/29, 27.5%), exon 21 (L858R Mutant (2/29, 6.8%)) in conjunctival SCCs. We also showed that the translocation of EGFR from the membrane into the cytoplasm was related to clinical activation of cancer, as correlations between EGFR cytoplasmic staining and final orbital exenteration, and between decreased EGFR membrane staining and PFS were noted. Although the number of cases examined was small, the expression of cytoplasmic staining of EGFR was weak but significantly different from membrane staining in the benign disease group. Our hypothesis is that as EGFR transitions from the membrane into the cytoplasm, malignant changes progress. In addition, a correlation between EGFR staining (focal or diffuse) and EGFR cytoplasmic staining was seen, and a higher score tended to be present in the diffuse EGFR staining group.

Intracellular transfer of EGFR in the group with diffuse staining may indicate progression, and although no statistical differences were observed in this study, significant findings may emerge by increasing the number of cases in the future.

In the past, especially in African countries, several studies on conjunctival SCCs and EGFR expression have been reported. They suggested a potential association with HPV [13, 14]. Other previous studies reported that post-translational modification can promote EGFR endocytosis and lysosomal degradation of EGFR, thereby ensuring termination of receptor signaling [15, 16].

In our cohort, expression and localization of EGFR and its association with prognosis were first reported in the Asian race. Additionally, intracellular translocation of EGFR from membrane staining to cytoplasm staining, likely by endocytosis, was associated with the percent of final orbital exenteration (cytoplasmic staining HR: 4.206, P < 0.036) and PFS (membrane staining HR: 0.237, P < 0.015) in our cohort. Regarding the difference in local changes in EGFR immunoreactivity in patients without EGFR expression in the tumor, we compared the Tis and Tadv groups according to AJCC T grading. A recent study showed that feedback regulatory loops can modulate growth factors and receptor tyrosine kinases such as EGFR to regulate cellular functions including abnormal states such as cancer [17]. Our study examined this phenomenon clinically and confirmed a pathological difference without changes in gene expression.

EGFR mutations in OSSN including invasive SCCs have not been examined in Asian patients. Since 2016, approximately 16,000 EGFR mutations in lung cancer had been registered in the COSMIC (the catalog of somatic mutations in cancer) database. Most (93%) are concentrated in the exon 18–21 region of the intracellular tyrosine kinase domain. The most frequent one is at codon 746 of exon 19. A deletion mutation is present at a site centered on five amino acids (ELREA) near amino acid 750, and a point mutation changes leucine to arginine (L858R) at codon 858 of exon 21 [18]. Shigematsu et al. in 2005 and Mitsudomi et al. in 2007 reported that EGFR mutations are common in Asians, females, non-smokers, and adenocarcinomas in lung cancer [19, 20]. Generally, when EGFR mutation occurs, the tyrosine kinase activity of EGFR at the ATP binding site is constantly active, even without growth factor. Cancer cell growth and survival depend on this pathway (oncogene addiction). EGFR TKIs competitively inhibit ATP binding in the kinase domain and suppress autophosphorylation of EGFR. Blockade of signal transmission has antitumor effects [21]. Previous reports of EGFR activating mutations (common mutations) described the frequency of exon 19 deletion mutations as 44.8% (2573/5741) and 39.8% for L858R mutations (2283/5731) in lung cancer [18, 22, 23].

EGFR mutations were examined to verify the effect of gefitinib on positive non-small cell lung carcinoma in two Phase III clinical trials from Japan. In the NEJ002 trial and the WJTOG3405 trial, gefitinib was the test treatment group; the standard treatment in the former was carboplatin + paclitaxel, and in the latter was cisplatin. In all studies, the gefitinib group showed superior PFS [24, 25]. In view of these findings in lung cancer in Asians, our findings regarding EGFR expression and mutations will provide further options for potential treatment of OSSN for pre- and post-surgical treatment.

The association of SCC with HPV was not confirmed because the number of cases was small. In addition, our results may not be accurate because we did not use multiplex PCR, which is currently the most suitable genotyping method [26].

Ours is the first report to show that differences in the expression form and mutations in EGFR in OSSN are associated with prognosis and treatment.

In an animal model, EGFR inhibition affected epithelial cell proliferation and stratification during corneal epithelial wound healing and may play a role in maintaining normal corneal epithelial thickness [27].

Gefitinib is an EGFR inhibitor and is the first approved molecular targeted therapy for cancer treatment in Japan [28]. Thus, understanding the pathological role of EGFR in OSSN and applying it to treatment are of great significance for seeking new treatment indications in OSSN including conjunctival SCCs. In this study, EGFR may translocate from the cell membrane into the cytoplasm. Tumor cells may transfer EGFR into the cytoplasm by endocytosis to avoid excessive signaling by the feedback system (Fig 5) [29]. Furthermore, in this study, the EGFR mutation was present in many patients with OSSN. This finding may suggest a course of treatment in the future. In addition, the method we used for identification of EGFR mutations was not general genotyping, but was a judgment of immunohistochemically stained sections. Although the sensitivity and specificity were high in a previous report, this is still a limitation [30].

This study has important limitations. First, regarding EGFR expression on the ocular surface, changes in benign diseases and age-related changes in normal tissues may not have been sufficiently investigated. Our study found that EGFR mutations were also present in conjunctival SCC in east Asians. However, we did not obtain results that correlated with the final prognosis. Further studies including further multi-institutional studies and an increase in the number of cases will be needed in the future. Another limitation is that double testing of formalin-fixed paraffin-embedded specimens and plasma with real-time PCR for detection of EGFR mutations is more common than IHC in actual clinical practice. According to the literature, both the sensitivity and specificity were satisfactory for these two types of mutations [30]. In addition, the size of our study cohort was small (n = 29), and the length of follow-up (less than 1 year in some patients) may not have been sufficient for long-term outcome analyses. Therefore, additional studies will be needed to corroborate our findings.

In conclusion, the results of this study indicate that EGFR is an active molecular target in the pathology of OSSN including SCC and is a prognostic factor. The finding also suggests that discovery of mutations may have important implications for future treatment options.

Supporting information

S1 File

(XLSX)

Click here for additional data file.^{(10.2KB, xlsx)}

Acknowledgments

We gratefully acknowledge the technical assistance of the Research Support Platform, Osaka City University Graduate School of Medicine and the Clinical Laboratory Department of Kobe Kaisei Hospital.

Data Availability

All relevant data are within the manuscript and Supporting Information files.

Funding Statement

None of the authors have any proprietary or financial interests to declare.

References

1.Lee GA, Hirst LW. Ocular surface squamous neoplasia. Surv Ophthalmol. 1995; 39: 429–450. 10.1016/s0039-6257(05)80054-2 [DOI] [PubMed] [Google Scholar]
2.Kiire CA, Stewart RMK, Srinivasan S, Heimann H, Kaye SB, Dhillon B. A prospective study of the incidence, associations and outcomes of ocular surface squamous neoplasia in the United Kingdom. Eye (Lond). 2018; 33: 283–294. [DOI] [PMC free article] [PubMed] [Google Scholar]
3.McClellan AJ, McClellan AL, Pezon CF, Karp CL, Feuer W, Galor A. Epidemiology of ocular surface squamous neoplasia in a veterans affairs population. Cornea. 2013; 32: 1354–1358. 10.1097/ICO.0b013e31829e3c80 [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Sun EC, Fears TR, Goedert JJ. Epidemiology of squamous cell conjunctival cancer. Cancer Epidemiol Biomarkers Prev. 1997; 6: 73‒77. [PubMed] [Google Scholar]
5.Scholz SL, Thomasen H, Reis H, et al. Frequent TERT promoter mutations in ocular surface squamous neoplasia. Invest Ophthalmol Vis Sci. 2015; 56: 5854‒5861. 10.1167/iovs.15-17469 [DOI] [PubMed] [Google Scholar]
6.Nagarajan P, El-Hadad C, Gruschkus SK, Ning J, Hudgens CW, Sagiv O, et al. PD-L1/PD1 Expression, Composition of Tumor-Associated Immune Infiltrate, and HPV Status in Conjunctival Squamous Cell Carcinoma. Invest Ophthalmol Vis Sci. 2019; 60: 2388‒2398. 10.1167/iovs.19-26894 [DOI] [PMC free article] [PubMed] [Google Scholar]
7.Le Tourneau C, Delord JP, Gonçalves A, Gavoille C, Dubot C, Isambert N, et al. Molecularly targeted therapy based on tumour molecular profiling versus conventional therapy for advanced cancer (SHIVA): a multicentre, open-label, proof-of-concept, randomised, controlled phase 2 trial. Lancet Oncol. 2015; 16: 1324‒1334. 10.1016/S1470-2045(15)00188-6 [DOI] [PubMed] [Google Scholar]
8.El Zaoui I, Bucher M, Rimoldi D, Nicolas M, Kaya G, Pescini Gobert R, et al. Conjunctival Melanoma Targeted Therapy: MAPK and PI3K/mTOR Pathways Inhibition. Invest Ophthalmol Vis Sci. 2019; 60(7): 2764‒2772. 10.1167/iovs.18-26508 [DOI] [PubMed] [Google Scholar]
9.Ciardiello F, Tortora G. A novel approach in the treatment of cancer: targeting the epidermal growth factor receptor. Clin Cancer Res. 2001; 7: 2958‒2970. [PubMed] [Google Scholar]
10.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: 2129‒2139. 10.1056/NEJMoa040938 [DOI] [PubMed] [Google Scholar]
11.Paez JG, Jänne 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: 1497‒1500. 10.1126/science.1099314 [DOI] [PubMed] [Google Scholar]
12.Cesano A. nCounter(®) PanCancer Immune Profiling Panel (NanoString Technologies, Inc., Seattle, WA). J Immunother Cancer. 2015; 3: 42 10.1186/s40425-015-0088-7 [DOI] [PMC free article] [PubMed] [Google Scholar]
13.Yu JJ, Fu P, Pink JJ, Dawson D, Wasman J, Orem J, et al. HPV infection and EGFR activation/alteration in HIV-infected East African patients with conjunctival carcinoma. PLoS One. 2010; 5: e10477 10.1371/journal.pone.0010477 [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Mwololo A, Nyagol J, Rogena E, Ochuk W, Kimani M, Onyango N, et al. Correlation of EGFR, pEGFR and p16INK4 expressions and high risk HPV infection in HIV/AIDS-related squamous cell carcinoma of conjunctiva. Infect Agent Cancer. 2014; 9: 7 10.1186/1750-9378-9-7 [DOI] [PMC free article] [PubMed] [Google Scholar]
15.Haglund K, Dikic I. The role of ubiquitylation in receptor endocytosis and endosomal sorting. J Cell Sci. 2012; 125: 265‒275. 10.1242/jcs.091280 [DOI] [PubMed] [Google Scholar]
16.Zhang X, Gureasko J, Shen K, Cole PA, Kuriyan J. An allosteric mechanism for activation of the kinase domain of epidermal growth factor receptor. Cell. 2006; 125: 1137‒1149. 10.1016/j.cell.2006.05.013 [DOI] [PubMed] [Google Scholar]
17.Avraham R, Yarden Y. Feedback regulation of EGFR signalling: decision making by early and delayed loops. Nat Rev Mol Cell Biol. 2011; 12(2): 104‒117. 10.1038/nrm3048 [DOI] [PubMed] [Google Scholar]
18.Kobayashi Y, Mitsudomi T. Not all epidermal growth factor receptor mutations in lung cancer are created equal: Perspectives for individualized treatment strategy. Cancer Sci. 2016; 107: 1179‒1186. 10.1111/cas.12996 [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Shigematsu H, Lin L, Takahashi T, Nomura M, Suzuki M, Wistuba II, et al. Clinical and biological features associated with epidermal growth factor receptor gene mutations in lung cancers. J Natl Cancer Inst. 2005; 97: 339‒346. 10.1093/jnci/dji055 [DOI] [PubMed] [Google Scholar]
20.Mitsudomi T, Yatabe Y. Mutations of the epidermal growth factor receptor gene and related genes as determinants of epidermal growth factor receptor tyrosine kinase inhibitors sensitivity in lung cancer. Cancer Sci. 2007; 98: 1817‒1824. 10.1111/j.1349-7006.2007.00607.x [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Yun CH, Mengwasser KE, Toms AV, Woo MS, Greulich H, Wong KK, et al. The T790M mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP. Proc Natl Acad Sci U S A. 2008; 105: 2070‒2075. 10.1073/pnas.0709662105 [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Kobayashi Y, Togashi Y, Yatabe Y, Mizuuchi H, Jangchul P, Kondo C, et al. EGFR Exon 18 Mutations in Lung Cancer: Molecular Predictors of Augmented Sensitivity to Afatinib or Neratinib as Compared with First- or Third-Generation TKIs. Clin Cancer Res. 2015; 21: 5305‒5313. 10.1158/1078-0432.CCR-15-1046 [DOI] [PubMed] [Google Scholar]
23.Wu JY, Yu CJ, Chang YC, Yang CH, Shih JY, Yang PC. Effectiveness of tyrosine kinase inhibitors on "uncommon" epidermal growth factor receptor mutations of unknown clinical significance in non-small cell lung cancer. Clin Cancer Res. 2011; 17: 3812‒3821. 10.1158/1078-0432.CCR-10-3408 [DOI] [PubMed] [Google Scholar]
24.Maemondo M, Inoue A, Kobayashi K, Sugawara S, Oizumi S, Isobe H, et al. Gefitinib or chemotherapy for non-small-cell lung cancer with mutated EGFR. N Engl J Med. 2010; 362: 2380‒2388. 10.1056/NEJMoa0909530 [DOI] [PubMed] [Google Scholar]
25.Mitsudomi T, Morita S, Yatabe Y, Negoro S, Okamoto I, Tsurutani J, et al. Gefitinib versus cisplatin plus docetaxel in patients with non-small-cell lung cancer harbouring mutations of the epidermal growth factor receptor (WJTOG3405): an open label, randomised phase 3 trial. Lancet Oncol. 2010; 11: 121‒128. 10.1016/S1470-2045(09)70364-X [DOI] [PubMed] [Google Scholar]
26.Nishiwaki M, Yamamoto T, Tone S, Murai T, Ohkawara T, Matsunami T, et al. Genotyping of human papillomaviruses by a novel one-step typing method with multiplex PCR and clinical applications. J Clin Microbiol. 2008; 46(4): 1161–1168. 10.1128/JCM.00793-07 [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Nakamura Y, Sotozono C, Kinoshita S. The epidermal growth factor receptor (EGFR): role in corneal wound healing and homeostasis. Exp Eye Res. 2001; 72: 511‒517. 10.1006/exer.2000.0979 [DOI] [PubMed] [Google Scholar]
28.Fukuoka M, Yano S, Giaccone G, Tamura T, Nakagawa K, Douillard JY, et al. Multi-institutional randomized phase II trial of gefitinib for previously treated patients with advanced non-small-cell lung cancer (The IDEAL 1 Trial) [corrected]. J Clin Oncol. 2003; 21: 2237‒2246. 10.1200/JCO.2003.10.038 [DOI] [PubMed] [Google Scholar]
29.Oldrini B, Hsieh WY, Erdjument-Bromage H, Codega P, Carro MS, Curiel-García A, et al. EGFR feedback-inhibition by Ran-binding protein 6 is disrupted in cancer. Nat Commun. 2017; 8: 2035 10.1038/s41467-017-02185-w [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Kane S, Wu J, Benedettini E, Li D, Reeves C, Innocenti G, et al. Mutation-specific antibodies for the detection of EGFR mutations in non-small-cell lung cancer. Clin Cancer Res. 2009; 15: 3023‒3028. 10.1158/1078-0432.CCR-08-2739 [DOI] [PubMed] [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0238120.r001

Decision Letter 0

Sanjoy Bhattacharya

20 May 2020

PONE-D-20-09283

Expression, intracellular localization, and mutation of EGFR in conjunctival squamous cell carcinoma associated with prognosis and treatment

PLOS ONE

Dear Dr. Tagami,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Reviewer #1: Partly

Reviewer #2: Partly

Reviewer #3: Partly

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: I Don't Know

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Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: This is an interesting study that evaluates EGFR status (intracellular localization and mutation spectrum) in conjunctival squamous carcinoma. Trying to fill a void in the basic science research on conjunctival SCC, the paper fails to adequately address the aims of the study.

Comments:

1. HPV status, role in tumorigenesis. The authors are right in their conclusion that the number of cases was small but the manner in which they assessed HPV infection needs revision. They reference to several studies on conjunctival SCC and HPV but the 2 papers they cite are on HIV positive patients of African descent. In the current era of understanding the role of high and low risk HPV in human tumorigenesis and differential testing for various HPV genotypes, the manner they chose to assess HPV, by IHC with a (probably) very broad HPV antibody lacks the desired specificity. The authors should consider either eliminating this part of the study or reevaluation of HPV status based on more recent literature pertinent to the immunocompetent subjects and in conjunction with employing more specific methods.

2. The authors use immunohistochemistry staining to draw conclusions on the intracellular distribution of EGFR and the translocation of EGFR from the membrane into the cytoplasm. In table 2 they show that all cases of invasive SCC and 14/15 cases of in situ carcinoma show various degrees (weak in the majority of cases) of cytoplasmic staining in combination with membranous staining (14/14 in invasive SCC, 14/15 in situ carcinoma). There is no correlation between the degree/intensity of membranous vs cytoplasmic staining. Assuming that cytoplasmic staining marks translocation of EGFR from the membrane into cytoplasm is not scientifically sound.

3. The authors claim that this is the first report to show that mutations in EGFR are associated with prognosis and treatment. They allude to the EGFR mutations in lung cancer and they show in table 3 the results of the 2 investigated mutations but there is no correlation of the 2 mutations with prognosis and treatment in ocular neoplasia.

4. The specificity of the 2 antibodies used for the 2 EGFR mutations is not addressed and no correlation with molecular studies identifying the same 2 mutations is done. However, the authors conclude that IHC can be used to identify EGFR mutations.

Reviewer #2: In the manuscript entitled " Expression, intracellular localization, and mutation of EGFR in conjunctival squamous cell carcinoma associated with prognosis and treatment” Sakai A. et al attempted to identify molecular localization of epidermal growth factor receptor (EGFR) and its role as novel biomarker for the management of conjunctival SCC. Authors used immunohistochemistry approach to evaluate EGFR expression and localization. They also used similar approach to visualize mutation associated with EGFR in SCC. Authors further implicated the translocation of EGFR from the membrane into the cytoplasm is prognostic marker for conjunctival squamous cell carcinoma. This is an interesting clinical study which focusses only east Asian population, however, a few major issues need to be addressed before the manuscript gets ready for the publication. The major concerns are as follows:

1. Authors have first found out that EGFR have focal and diffuse staining in patients. This is really an interesting observation however, authors later never looked for its correlation with localization or mutation of EGFR in patients.

2. In figure 1A&B, authors showed representative images of local and diffuse EGFR staining. It appears that images of EGFR staining in both figures have different magnification. Similarly figure 1E captured at different magnification compared to figure 1C&D. Figure 2 have similar issue where image D have different magnification compared to rest of the images. Authors need to make sure representative images were captured at same magnification for better comparison. Authors also need add scale bar on all the microscopy images.

3. In figure 1A hematoxylin-eosin-stained section in inset looks little different from EGFR stained image. Authors also need to make sure that the image in H&E inset properly corresponds to EGFR stained image.

4. In present manuscript authors also evaluated EGFR mutation using immunohistochemistry. They observed EGFR E746-A750 del and EGFR L858R expression in patients. Authors need to include this parameter for univariate correlation analysis with PFS and orbital exenteration. Also, it will be interesting to see the effect of these mutation on the localization of EGFR in tumor cell.

5. Authors also need to perform multivariate analysis for clinical correlation for both membrane and cytoplasmic EGFR localization in patients.

6. References in the manuscript have different fonts and wrongly numbered. Authors need to use endnote or reference manager to add the references.

Reviewer #3: In the submitted manuscript, Sakai et al evaluated the expression, localization, and mutation of EGFR in conjunctival squamous cell carcinoma. Using immunohistochemistry, they observed that all 29 samples were EGFR positive and classified its expression into membrane, cytoplasm, focal, and diffuse. Eight samples presented the E746-A750 del mutation while 2 samples the L858R point mutation. They also found a correlation between EGFR cytoplasmic staining and orbital exenteration, and between decreased EGFR membrane staining and progression-free survival. Although, the role of EGFR has been extensively study in many different cancer types, little is known about EGFR in conjunctival squamous cell carcinoma. The current study brings valuable information to the field and is on a topic of relevance. However, some concerns must be addressed before further consideration.

Specific comments

1. The result section should be written in more detail for easier reading.

2. Line 154: “Analysis of EGFR intracellular staining patterns showed scores of 1.72 for membrane staining and 1.48 for cytoplasmic staining (Figure 1)”. This result is found in Table 2 not in figure 1.

3. Table 2: Revise statistics. My calculation doesn’t match the average shown in the table.

4. Is there any difference in EGFR expression localization (membrane or cytoplasm) between focal and diffuse staining?

5. Line 156: “Seven benign conjunctival lesions were partially weak positive in conjunctival squamous epithelial cells near basement membrane”. Specify the total number of benign samples evaluated (7 out of 7?). It would be interesting to show the score analysis of benign samples to compare to tumor samples.

6. Line 157: “We found no significant changes between Tis (in situ) and Tadv (SCC advanced cases) (Table 2)”. Specify what changes.

7. Line 159: Include more information regarding EGFR E746-A750 del and EGFR L858R expression like diffuse/focal staining, membrane/cytoplasmic score. Is there any correlation between EGFR mutation and clinical data (orbital exenteration/PFS)?

8. Table 1: EGFR expression in tumors - diffuse staining (8) and focal staining (19). Are these numbers correct, total samples 27?

9. Figure 1 and 2: When comparing images use the same magnification. Verify if scale bars are correct.

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Reviewer #3: No

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PLoS One. 2020 Aug 24;15(8):e0238120. doi: 10.1371/journal.pone.0238120.r002

Author response to Decision Letter 0

12 Jun 2020

Response to reviewers

Reviewer #1: Thank you for your valuable comments.

Our responses to your comments are below.

Comments:

#This was a very meaningful suggestion. I completely agree with the reviewer. References to HPV in the conclusion section have been deleted.

L244 In the Discussion, we added the following:

#Thank you for your valuable suggestions. For this phenomenon, we further compared our cases with cases of benign conjunctival mass lesions. Although the number was small (n = 7), the following results were obtained.

Staining of the cytoplasm was significantly different between the benign disease group and malignant cases, which was a new finding for all conjunctival tumors. This information has been added to the Results and Discussion.

L158- seven benign conjunctival lesions ( three pinguecula, three pterygium, one dermoid cyst) were partially weak positive in conjunctival squamous epithelial cells , especially cell surface membranes(Figure 2).In addition, cytoplasmic staining are only one case. Benign cases showed scores of 1.28 for membrane staining and 0.14 for cytoplasmic staining, compared with conjunctival Scc cases, Cytoplasmic staining was statistically significant changes ( P<0.01) (Table.2).

L200- Although the number of cases examined was small, the expression of cytoplasmic staining of EGFR was significantly weak in the benign disease group. The hypothesis might be that the EGFR transition from membrane surface into cytoplasm will progress as it becomes more malignancy changes.

#Thank you very much for pointing this out.

The Cox regression model was used to examine the relationship between EGFR mutations and long-term prognosis including orbital exenteration and PFS. EGFR mutations were not significantly correlated with final orbital exenteration or PFS (Tables 4 and 5). The small number of cases may be the reason why no significant difference was found. Examination of these cases suggested that mutation-positive cases also tended to have slightly advanced cancer, but no statistically significant difference was found. The following was added to the conclusion.

L264 Our study found that EGFR mutations were also found in the conjunctival Scc, but we could not obtain the results that would correlate with the final prognosis. It seems likely that further studies including further multi-institutions and an increase in the number of cases will be expected in the future.

#This is a reasonable question.

Certainly, double testing of formalin-fixed paraffin-embedded tissue and plasma with real-time PCR for detection of EGFR mutations is more common than IHC in the actual clinical setting. According to the literature, both sensitivity and specificity seemed to be satisfactory for these two types of mutations. Reference No. 30 was added. We also described this as a limitation.

30. Kane S, Wu J, Benedettini E, Li D, Reeves C, Innocenti G, Wetzel R, Crosby K, Becker A, Ferrante M, Cheung WC, Hong X, Chirieac LR, Sholl LM, Haack H, Smith BL, Polakiewicz RD, Tan Y, Gu TL, Loda M, Zhou X, Comb MJ.. Mutation-specific antibodies for the detection of EGFR mutations in non -small-cell lung cancer. Clin Cancer Res 2009;15:3023-3028.

Reviewer #2:

Thank you for your valuable comments.

Our responses to your comments are below.

#This is a very meaningful suggestion.

We also realized that this point was a problem, and thus, we reviewed the scoring for each of the two groups. Then, we performed statistical analysis with linear regression calculation. Unfortunately, the cytoplasmic group did not show a statistically significant difference, but the diffuse EGFR group tended to have a higher cytoplasmic score (P = 0.077). The membrane group did not show a significant difference or tendency for a higher score (P = 0.214). We added this to the Results and also mentioned it in the Discussion.

L164

The correlation between EGFR stating (focal or diffuse) and EGFR localization (cytoplasmic staining group ) did not show a statistically significant difference, but the diffuse EGFR group tended to have a higher score(p=0.077). The correlation between EGFR stating (focal or diffuse) and EGFR localization (membrane group) was no significant difference and no tendency (p=0.214).

L200

Although the number of cases examined was small, the expression of cytoplasmic staining of EGFR was weak but significantly different from membrane staining. Our hypothesis is that as EGFR transitions from the membrane into the cytoplasm, malignant changes progress. In addition, a correlation between EGFR staining (focal or diffuse) and EGFR cytoplasmic staining was seen, and a higher score tended to be present in the diffuse EGFR staining group.

Although the difference was not significant in this study, intracellular transfer of EGFR in the group with diffuse staining may indicate progression, and significant findings may emerge by increasing the number of cases in the future.

L173 The relationship between EGFR mutation and EGFR stating (focal or diffuse) was determined using univariate linear regression analysis with making corrections based on age(p=0.559)

#Thank you for your valuable suggestions.

We added scale bars and corrected the figures as you suggested (Figure 1 and Figure 2). In one panel, the HE-stained section does not match, but the same region is photographed and shown. We are sorry for the incompleteness.

#Thank you for your valuable suggestions.

L180 The Cox regression model was used to examine and analyze the relationship between long-term prognosis including orbital exenteration and PFS and the clinicopathological status, EGFR staining pattern, and EGFR mutation.

L187 The EGFR mutation was not significantly correlated with final orbital exenteration or PFS (Tables 4 and 5). The small number of cases may be the reason why no significant difference was found.

5. Authors also need to perform multivariate analysis for clinical correlation for both membrane and cytoplasmic EGFR localization in patients.

#Thank you very much for pointing this out.

We also consulted with statisticians at our university, and discussed the number of cases. Because the number of parameters was small and difficult to evaluate with multivariate analysis, we decided not to perform further analysis at this time. We appreciate your understanding.

6. References in the manuscript have different fonts and wrongly numbered. Authors need to use endnote or reference manager to add the references.

#Thank you very much for pointing this out.

We will correct the incorrect parts. Although we have requested funding for Endnote, it cannot be purchased immediately. Thank you very much.

reviewer3

Thank you for your valuable comments.

Our responses to your comments are below.

1. The result section should be written in more detail for easier reading.

#Thank you for your valuable suggestions.

We added the text below to the Results section.

L156-

No significant difference was found between carcinoma in situ (Tis) and invasive carcinoma (Tadv) (Table 2). No significant difference was found in the score depending on the stage. On the other hand, seven benign conjunctival lesions (three pinguecula, three pterygium, one dermoid cyst) showed partial weak positive staining in conjunctival squamous epithelial cells, especially on the cell membrane (Figure 2A). EGFR expression in colon cancer for positive control (Figure.2B).　In addition, cytoplasmic staining was seen in only one case. Benign cases showed scores of 1.28 for membrane staining and 0.14 for cytoplasmic staining. Cytoplasmic staining patterns were significantly different in benign compared to SCC cases (P < 0.01) (Table 2). The correlation between EGFR staining (focal or diffuse) and EGFR localization (cytoplasmic staining group) was not significantly different, but the diffuse EGFR group tended to have a higher score (P = 0.077). No correlation was found between EGFR staining (focal or diffuse) and EGFR localization (membrane group), and no apparent tendency was observed (P = 0.214).

L170

The mutation at exon 19, EGFR E7446-A750 del, was confirmed in 8/29 (27.5%) cases, and that at exon 21, EGFR L858R point mutation, was confirmed in 2/29 (6.8%) cases with IHC (Table 3). The relationship between EGFR mutation and EGFR staining (focal or diffuse) was determined using univariate linear regression analysis with correction for age (P = 0.559).

L180

COX regression model was used to examine and analysis among clinicopathological status, EGFR staining pattern, and EGFR mutation for long-term prognosis.

L187

In addition, the EGFR mutation was not significantly correlation with final orbital exenteration and PFS(Table 5).

#Thank you for pointing this out.

L157 We corrected the text from Figure 1 to Table 2.

3. Table 2: Revise statistics. My calculation doesn’t match the average shown in the table.

# We are sorry for this mistake.

P21 Table 2. We recalculated and now show the correct value in Table 2.

4. Is there any difference in EGFR expression localization (membrane or cytoplasm) between focal and diffuse staining?

#This is a very meaningful suggestion.

We also realized that this point was a problem, and again reviewed the scoring for each of the two groups. Then, we performed statistical analysis with linear regression calculation. Unfortunately, the cytoplasmic group did not show a statistically significant difference, but the diffuse EGFR group tended to have a higher score (P = 0.077). The membrane group did not show a significant difference or tendency (P = 0.214). We added this to the Results and also mentioned it in the Discussion.

L159-169

L201 Although the number of cases examined was small, the expression of cytoplasmic staining of EGFR was weak but significantly different from membrane staining. Our hypothesis is that as EGFR transitions from the membrane into the cytoplasm, malignant changes progress. In addition, a correlation between EGFR staining (focal or diffuse) and EGFR cytoplasmic staining was seen, and a higher score tended to be present in the diffuse EGFR staining group.

#This was a very meaningful suggestion. Thank you very much.

The staining of the cytoplasm was significantly different between the benign disease group and malignant cases, which was a new finding for conjunctival tumors. This information was added to the Results and Discussion.

L159- seven benign conjunctival lesions ( three pinguecula, three pterygium, one dermoid cyst) were partially weak positive in conjunctival squamous epithelial cells , especially cell surface membranes(Figure 2).In addition, cytoplasmic staining are only one case. Benign cases showed scores of 1.28 for membrane staining and 0.14 for cytoplasmic staining, compared with conjunctival Scc cases, Cytoplasmic staining was statistically significant changes ( P<0.01) (Table.2).

L201- Although the number of cases examined was small, the expression of cytoplasmic staining of EGFR was significantly weak in the benign disease group. The hypothesis might be that the EGFR transition from membrane surface into cytoplasm will progress as it becomes more malignancy changes.

6. Line 157: “We found no significant changes between Tis (in situ) and Tadv (SCC advanced cases) (Table 2)”. Specify what changes.

We deleted the original sentence and added the following sentence.

L156 No statistical difference was found between Tis and Tadv in the intensity of immunostaining in staining patterns.

#Thank you for your valuable suggestions.

The Cox regression model was used to examine and analyze the relationship between long-term prognosis including orbital exenteration and PFS and the clinicopathological status, EGFR staining pattern, and EGFR mutation. The EGFR mutation was not significantly correlated with final orbital exenteration or PFS (Tables 4 and 5). The small number of cases may be the reason why no significant difference was found.

8. Table 1: EGFR expression in tumors - diffuse staining (8) and focal staining (19). Are these numbers correct, total samples 27?

# We are sorry for this mistake.

P19 Table 1. The value was recalculated and described correctly in Table 1.

9. Figure 1 and 2: When comparing images use the same magnification. Verify if scale bars are correct.

#Thank you for your valuable suggestions.

We inserted scale bars and corrected the figures (Figure 1 and Figure 2).

PLoS One. doi: 10.1371/journal.pone.0238120.r003

Decision Letter 1

Sanjoy Bhattacharya

15 Jul 2020

PONE-D-20-09283R1

Expression, intracellular localization, and mutation of EGFR in conjunctival squamous cell carcinoma and the association with prognosis and treatment

PLOS ONE

Dear Dr.Tagami,

A learned reviewer has raised a number of critical issues that need to be satisfactorily addressed by incorporating appropriate changes in the manuscript.

Please submit your revised manuscript by Aug 29 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Sanjoy Bhattacharya

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: (No Response)

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

**********

6. Review Comments to the Author

Reviewer #1: Comments have been addressed. The manuscript has spelling and minor grammatical errors that need to be fixed.

Reviewer #2: (No Response)

Reviewer #3: In this revised manuscript, the authors have addressed some comments raised from my previous review report. However, there are several major concerns that need to be addressed before further consideration.

1. A major revision of language and grammar is necessary for any further consideration. There are several typos, missing spaces between words, and grammatically incorrect sentences (especially in the new text added after revision) that make it hard to read. Some examples are:

Line 161 - Benign cases showed scores of 1.28 for membrane staining and 0.14 for cytoplasmic staining, compared with conjunctival Scc cases, Cytoplasmic staining was statistically significant changes

Line 166 - The correlation between EGFR stating (focal or diffuse) and EGFR localization (membrane group) was no significant difference and tendency

Line 180 - COX regression model was used to examine and analysis among clinicopathological status, EGFR staining pattern, and EGFR mutation for long-term prognosis including orbital exenteration and.PFS.

Line 188 - In addition, the EGFR 188 mutation was not significantly correlation with final orbital exenteration and PFS(Table 4 and 5).

2. Line 206 – “There was no significant difference in this study”. Specify what had no significant difference. The whole study or a specific result?

3. Line 160, the sentence “EGFR expression in colon cancer for positive control” is out of context. What is the relevance of this information to the results?

4. Line 164-167 – Regarding the correlation between EGFR staining (focal or diffuse) and EGFR localization (membrane or cytoplasm), it would be interesting to have a table showing the number of diffuse and focal staining samples in the membrane, and cytoplasm group. Between the slides with EGFR membrane localization, how many samples had focal staining and how many had diffuse staining? Add the same information for EGFR cytoplasm localization.

5. In table 3, add information (number of samples) regarding focal, diffuse, membrane, and cytoplasmic staining for each mutation.

6. In figure 3C, the EGFR L858R staining seems to be localized in the nucleus. Do you have an explanation for that?

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2020 Aug 24;15(8):e0238120. doi: 10.1371/journal.pone.0238120.r004

Author response to Decision Letter 1

21 Jul 2020

Response to reviewers

Reviewer #1: Thank you for your valuable comments. We submitted the revised version for proofreading by a native English-speaking science editor.

Reviewer #3

Thank you for your valuable comments. Our responses are below.

Line 161 - Benign cases showed scores of 1.28 for membrane staining and 0.14 for cytoplasmic staining, compared with conjunctival Scc cases, Cytoplasmic staining was statistically significant changes

Line 166 - The correlation between EGFR stating (focal or diffuse) and EGFR localization (membrane group) was no significant difference and tendency

Line 188 - In addition, the EGFR 188 mutation was not significantly correlation with final orbital exenteration and PFS(Table 4 and 5).

We submitted the revised version for proofreading by a native English-speaking science editor.

2. Line 206 – “There was no significant difference in this study”. Specify what had no significant difference. The whole study or a specific result?

Only the results of this statement are mentioned. We corrected the sentence as shown below.

L170“Intracellular transfer of EGFR in the group with diffuse staining may indicate progression, and although no statistical differences were observed in this study, significant findings may emerge by increasing the number of cases in the future.”

3. Line 160, the sentence “EGFR expression in colon cancer for positive control” is out of context. What is the relevance of this information to the results?

We corrected the sentence as shown below. The location was changed to line 163 to suit the context. Accordingly, we changed the location of the Figure 2 citation in the text.

We created a new Table 3 to address your comment.

Table3. EGFR staining and Localization

EGFR Focal(N=21) EGFR Diffuse（N=8) P

Cell Membrane 1.8±0.9

1.5±0.9 0.38

Cell cytoplasmic 1.3±0.6 1.8±0.8 0.12

p value based on the non-paired t-test.

5. In table 3, add information (number of samples) regarding focal, diffuse, membrane, and cytoplasmic staining for each mutation.

We created a new Table 4 to hold this information.

Table 4. Summary of EGFR E746-A750 del and EGFR L858R point mutations

Mutation N = 29 (%) Age (y) Sex (M/F) T stage EGFR staining patterns (diffuse/focal) EGFR localization score (membrane/cytoplasmic)

Exon 19 EGFR E746-A750 del (N=8) 8/29 (27.5%) 75.8 3/5 T3: 4

T2: 2

Tis: 2 2/6 1.6/1.5

Exon 21 EGFR L858R point mutation (N=3) 2/29 (6.8%) 73.0 1/1 T3: 1

Tis: 1 1/2 1/3

M: Male, F: Female

6. In figure 3C, the EGFR L858R staining seems to be localized in the nucleus. Do you have an explanation for that?

Thank you for this advice. We agree that regarding the dyeability of EGFR, it is important whether or not the cell membrane is stained. It looks like it stains the nucleus, but we are unsure of the exact reason. We believe that it differs from the simple background because only the nuclei of squamous cell carcinoma are stained.

There are the following documents. Whether or not this is meant is not convinced by this paper alone.

The receptor tyrosine kinases (RTK) of the epidermal growth factor receptor (EGFR) superfamily　in the membrane can translocate to the nucleus through different mechanisms. Nuclear RTKs regulate a variety of cellular functions, such as cell proliferation, DNA damage repair, and signal transduction, both in normal tissues and in human cancer cell. In addition, they play important roles in determining cancer response to cancer therapy.（Shao-Chun Wang et al. DOI: 10.1158/1078-0432.CCR-08-2813 2009）

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PLoS One. doi: 10.1371/journal.pone.0238120.r005

Decision Letter 2

Sanjoy Bhattacharya

11 Aug 2020

Expression, intracellular localization, and mutation of EGFR in conjunctival squamous cell carcinoma and the association with prognosis and treatment

PONE-D-20-09283R2

Dear Dr. Tagami,

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Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

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

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

Reviewer #2: (No Response)

Reviewer #3: Yes

**********

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

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Reviewer #2: (No Response)

Reviewer #3: (No Response)

**********

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PLoS One. doi: 10.1371/journal.pone.0238120.r006

Acceptance letter

Sanjoy Bhattacharya

13 Aug 2020

PONE-D-20-09283R2

Expression, intracellular localization, and mutation of EGFR in conjunctival squamous cell carcinoma and the association with prognosis and treatment

Dear Dr. Tagami:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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on behalf of

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[pone.0238120.ref001] 1.Lee GA, Hirst LW. Ocular surface squamous neoplasia. Surv Ophthalmol. 1995; 39: 429–450. 10.1016/s0039-6257(05)80054-2 [DOI] [PubMed] [Google Scholar]

[pone.0238120.ref002] 2.Kiire CA, Stewart RMK, Srinivasan S, Heimann H, Kaye SB, Dhillon B. A prospective study of the incidence, associations and outcomes of ocular surface squamous neoplasia in the United Kingdom. Eye (Lond). 2018; 33: 283–294. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0238120.ref003] 3.McClellan AJ, McClellan AL, Pezon CF, Karp CL, Feuer W, Galor A. Epidemiology of ocular surface squamous neoplasia in a veterans affairs population. Cornea. 2013; 32: 1354–1358. 10.1097/ICO.0b013e31829e3c80 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0238120.ref004] 4.Sun EC, Fears TR, Goedert JJ. Epidemiology of squamous cell conjunctival cancer. Cancer Epidemiol Biomarkers Prev. 1997; 6: 73‒77. [PubMed] [Google Scholar]

[pone.0238120.ref005] 5.Scholz SL, Thomasen H, Reis H, et al. Frequent TERT promoter mutations in ocular surface squamous neoplasia. Invest Ophthalmol Vis Sci. 2015; 56: 5854‒5861. 10.1167/iovs.15-17469 [DOI] [PubMed] [Google Scholar]

[pone.0238120.ref006] 6.Nagarajan P, El-Hadad C, Gruschkus SK, Ning J, Hudgens CW, Sagiv O, et al. PD-L1/PD1 Expression, Composition of Tumor-Associated Immune Infiltrate, and HPV Status in Conjunctival Squamous Cell Carcinoma. Invest Ophthalmol Vis Sci. 2019; 60: 2388‒2398. 10.1167/iovs.19-26894 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0238120.ref007] 7.Le Tourneau C, Delord JP, Gonçalves A, Gavoille C, Dubot C, Isambert N, et al. Molecularly targeted therapy based on tumour molecular profiling versus conventional therapy for advanced cancer (SHIVA): a multicentre, open-label, proof-of-concept, randomised, controlled phase 2 trial. Lancet Oncol. 2015; 16: 1324‒1334. 10.1016/S1470-2045(15)00188-6 [DOI] [PubMed] [Google Scholar]

[pone.0238120.ref008] 8.El Zaoui I, Bucher M, Rimoldi D, Nicolas M, Kaya G, Pescini Gobert R, et al. Conjunctival Melanoma Targeted Therapy: MAPK and PI3K/mTOR Pathways Inhibition. Invest Ophthalmol Vis Sci. 2019; 60(7): 2764‒2772. 10.1167/iovs.18-26508 [DOI] [PubMed] [Google Scholar]

[pone.0238120.ref009] 9.Ciardiello F, Tortora G. A novel approach in the treatment of cancer: targeting the epidermal growth factor receptor. Clin Cancer Res. 2001; 7: 2958‒2970. [PubMed] [Google Scholar]

[pone.0238120.ref010] 10.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: 2129‒2139. 10.1056/NEJMoa040938 [DOI] [PubMed] [Google Scholar]

[pone.0238120.ref011] 11.Paez JG, Jänne 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: 1497‒1500. 10.1126/science.1099314 [DOI] [PubMed] [Google Scholar]

[pone.0238120.ref012] 12.Cesano A. nCounter(®) PanCancer Immune Profiling Panel (NanoString Technologies, Inc., Seattle, WA). J Immunother Cancer. 2015; 3: 42 10.1186/s40425-015-0088-7 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0238120.ref013] 13.Yu JJ, Fu P, Pink JJ, Dawson D, Wasman J, Orem J, et al. HPV infection and EGFR activation/alteration in HIV-infected East African patients with conjunctival carcinoma. PLoS One. 2010; 5: e10477 10.1371/journal.pone.0010477 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0238120.ref014] 14.Mwololo A, Nyagol J, Rogena E, Ochuk W, Kimani M, Onyango N, et al. Correlation of EGFR, pEGFR and p16INK4 expressions and high risk HPV infection in HIV/AIDS-related squamous cell carcinoma of conjunctiva. Infect Agent Cancer. 2014; 9: 7 10.1186/1750-9378-9-7 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0238120.ref015] 15.Haglund K, Dikic I. The role of ubiquitylation in receptor endocytosis and endosomal sorting. J Cell Sci. 2012; 125: 265‒275. 10.1242/jcs.091280 [DOI] [PubMed] [Google Scholar]

[pone.0238120.ref016] 16.Zhang X, Gureasko J, Shen K, Cole PA, Kuriyan J. An allosteric mechanism for activation of the kinase domain of epidermal growth factor receptor. Cell. 2006; 125: 1137‒1149. 10.1016/j.cell.2006.05.013 [DOI] [PubMed] [Google Scholar]

[pone.0238120.ref017] 17.Avraham R, Yarden Y. Feedback regulation of EGFR signalling: decision making by early and delayed loops. Nat Rev Mol Cell Biol. 2011; 12(2): 104‒117. 10.1038/nrm3048 [DOI] [PubMed] [Google Scholar]

[pone.0238120.ref018] 18.Kobayashi Y, Mitsudomi T. Not all epidermal growth factor receptor mutations in lung cancer are created equal: Perspectives for individualized treatment strategy. Cancer Sci. 2016; 107: 1179‒1186. 10.1111/cas.12996 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0238120.ref019] 19.Shigematsu H, Lin L, Takahashi T, Nomura M, Suzuki M, Wistuba II, et al. Clinical and biological features associated with epidermal growth factor receptor gene mutations in lung cancers. J Natl Cancer Inst. 2005; 97: 339‒346. 10.1093/jnci/dji055 [DOI] [PubMed] [Google Scholar]

[pone.0238120.ref020] 20.Mitsudomi T, Yatabe Y. Mutations of the epidermal growth factor receptor gene and related genes as determinants of epidermal growth factor receptor tyrosine kinase inhibitors sensitivity in lung cancer. Cancer Sci. 2007; 98: 1817‒1824. 10.1111/j.1349-7006.2007.00607.x [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0238120.ref021] 21.Yun CH, Mengwasser KE, Toms AV, Woo MS, Greulich H, Wong KK, et al. The T790M mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP. Proc Natl Acad Sci U S A. 2008; 105: 2070‒2075. 10.1073/pnas.0709662105 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0238120.ref022] 22.Kobayashi Y, Togashi Y, Yatabe Y, Mizuuchi H, Jangchul P, Kondo C, et al. EGFR Exon 18 Mutations in Lung Cancer: Molecular Predictors of Augmented Sensitivity to Afatinib or Neratinib as Compared with First- or Third-Generation TKIs. Clin Cancer Res. 2015; 21: 5305‒5313. 10.1158/1078-0432.CCR-15-1046 [DOI] [PubMed] [Google Scholar]

[pone.0238120.ref023] 23.Wu JY, Yu CJ, Chang YC, Yang CH, Shih JY, Yang PC. Effectiveness of tyrosine kinase inhibitors on "uncommon" epidermal growth factor receptor mutations of unknown clinical significance in non-small cell lung cancer. Clin Cancer Res. 2011; 17: 3812‒3821. 10.1158/1078-0432.CCR-10-3408 [DOI] [PubMed] [Google Scholar]

[pone.0238120.ref024] 24.Maemondo M, Inoue A, Kobayashi K, Sugawara S, Oizumi S, Isobe H, et al. Gefitinib or chemotherapy for non-small-cell lung cancer with mutated EGFR. N Engl J Med. 2010; 362: 2380‒2388. 10.1056/NEJMoa0909530 [DOI] [PubMed] [Google Scholar]

[pone.0238120.ref025] 25.Mitsudomi T, Morita S, Yatabe Y, Negoro S, Okamoto I, Tsurutani J, et al. Gefitinib versus cisplatin plus docetaxel in patients with non-small-cell lung cancer harbouring mutations of the epidermal growth factor receptor (WJTOG3405): an open label, randomised phase 3 trial. Lancet Oncol. 2010; 11: 121‒128. 10.1016/S1470-2045(09)70364-X [DOI] [PubMed] [Google Scholar]

[pone.0238120.ref026] 26.Nishiwaki M, Yamamoto T, Tone S, Murai T, Ohkawara T, Matsunami T, et al. Genotyping of human papillomaviruses by a novel one-step typing method with multiplex PCR and clinical applications. J Clin Microbiol. 2008; 46(4): 1161–1168. 10.1128/JCM.00793-07 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0238120.ref027] 27.Nakamura Y, Sotozono C, Kinoshita S. The epidermal growth factor receptor (EGFR): role in corneal wound healing and homeostasis. Exp Eye Res. 2001; 72: 511‒517. 10.1006/exer.2000.0979 [DOI] [PubMed] [Google Scholar]

[pone.0238120.ref028] 28.Fukuoka M, Yano S, Giaccone G, Tamura T, Nakagawa K, Douillard JY, et al. Multi-institutional randomized phase II trial of gefitinib for previously treated patients with advanced non-small-cell lung cancer (The IDEAL 1 Trial) [corrected]. J Clin Oncol. 2003; 21: 2237‒2246. 10.1200/JCO.2003.10.038 [DOI] [PubMed] [Google Scholar]

[pone.0238120.ref029] 29.Oldrini B, Hsieh WY, Erdjument-Bromage H, Codega P, Carro MS, Curiel-García A, et al. EGFR feedback-inhibition by Ran-binding protein 6 is disrupted in cancer. Nat Commun. 2017; 8: 2035 10.1038/s41467-017-02185-w [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0238120.ref030] 30.Kane S, Wu J, Benedettini E, Li D, Reeves C, Innocenti G, et al. Mutation-specific antibodies for the detection of EGFR mutations in non-small-cell lung cancer. Clin Cancer Res. 2009; 15: 3023‒3028. 10.1158/1078-0432.CCR-08-2739 [DOI] [PubMed] [Google Scholar]

PERMALINK

Expression, intracellular localization, and mutation of EGFR in conjunctival squamous cell carcinoma and the association with prognosis and treatment

Atsushi Sakai

Mizuki Tagami

Anna Kakehashi

Atsuko Katsuyama-Yoshikawa

Norihiko Misawa

Hideki Wanibuchi

Atsushi Azumi

Shigeru Honda

Roles

Abstract

Purpose

Methods

Results

Conclusions

Introduction

Materials and methods

Selection of cases and collation of clinicopathologic data

Immunohistochemistry (IHC)

Image analysis

EGFR expression in tumors

Statistical analysis

Results

Table 1. Clinicopathologic findings of 29 cases of conjunctival squamous cell carcinoma.

Fig 1. EGFR expression in conjunctival SCC.

Table 2. Staining patterns of EGFR.

Fig 2.

Table 3. EGFR staining and localization.

Fig 3. EGFR mutation-specific expression in conjunctival SCC.

Table 4. Summary of EGFR E746-A750 del and EGFR L858R point mutations.

Fig 4. For EGFR expression in tumors, we compared carcinoma in situ (Tis) and invasive carcinoma (Tadv) groups according to AJCC T grading (n-4, 4).

Table 5. Relationship between orbital exenteration and clinicopathologic and molecular factors.

Table 6. Relationship between PFS and clinicopathologic and molecular factors.

Discussion

Fig 5. Schematic of movement of EGFR into the cytoplasm by endocytosis to avoid excessive signaling and for recycling.

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Sanjoy Bhattacharya

Roles

Author response to Decision Letter 0

Decision Letter 1

Sanjoy Bhattacharya

Roles

Author response to Decision Letter 1

Decision Letter 2

Sanjoy Bhattacharya

Roles

Acceptance letter

Sanjoy Bhattacharya

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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