Epidermal growth factor receptor expression is ... - Wiley Online Library

BJD

British Journal of Dermatology

T R AN SLA T IO NA L RE SE AR CH

Epidermal growth factor receptor expression is associated with poor outcome in cutaneous squamous cell carcinoma Santos-Briz,1,2 A. Castellanos-Martın,2,4 E. Ferna ~ueto,1,2 E. Carden ~oso,3 J.L. Garcıa,2,4 A. ndez-Lo pez,1,2 J. Can 2,4 2,4 1,2 mez, J. Pe rez-Losada and C. Roma n-Curto A. Blanco Go 1

Departamento de Dermatologıa and 2Instituto de Investigacion Biomedica de Salamanca (IBSAL), Hospital Universitario de Salamanca, Paseo de San Vicente, 58-182 37007 Salamanca, Spain 3 Departamento de Dermatologıa, Hospital Virgen de la Concha, Avenida de Requejo, Zamora, Spain 4 Instituto de Biologıa Molecular y Celular del Cancer (IBMCC), Universidad de Salamanca/CSIC, Campus Miguel de Unamuno, s/n. 37007 Salamanca, Spain Linked Comment: Griewank. Br J Dermatol 2017; 176:1126–1127

Summary Correspondence Javier Ca~nueto. E-mail: [email protected]

Accepted for publication 8 August 2016

Funding sources J.P.-L. was partially supported by FEDER and MICINN (PLE2009-119, SAF2014-56989-R), Instituto de Salud Carlos III (PI07/0057, PI10/00328, PIE14/00066), Junta de Castilla y Leon (SAN673/SA26/08, SAN126/SA66/ 09, SA078A09, CSI034U13, BIO/SA31/15), IBSAL (IBY15/00003), the ‘Eugenio Rodrıguez Pascual’, the ‘Fundacion Inbiomed’ (Instituto Oncologico Obra Social de la Caja Guipozcoa-San Sebastian, Kutxa) and the ‘Fundacion Sandra Ibarra de Solidaridad frente al Cancer’. C.R.-C. is funded by Q3718001E (2009-2010) and GRS 612/A/11 (2011-2012) and ‘the Fundacion Eugenio Rodrıguez Pascual’. A.C.-M. was supported by FIS (PI07/0057) and MICINN (PLE2009-119).

Conflicts of interest

Background Cutaneous squamous cell carcinoma (CSCC) is the second most frequent cancer in humans after basal cell carcinoma, and its incidence is dramatically rising. CSCC is rarely problematic, but given its high frequency, the absolute number of complicated cases is also high. It is necessary to identify molecular markers in order to recognize those CSCCs with poor prognosis. There is controversy concerning the role of epidermal growth factor receptor (EGFR) as a marker of prognosis in CSCC. In addition, EGFR-targeted therapies have emerged in recent years and a better understanding of the role of EGFR in CSCC may be of help for some patients in predicting prognosis and guiding curative management. Objectives To evaluate the role of EGFR as a prognostic factor in CSCC. Methods We evaluated clinical and histopathological features, including events of poor clinical evolution, in a series of 94 cases of CSCC. We also analysed EGFR expression by immunohistochemistry, fluorescent in situ hybridization and quantitative polymerase chain reaction. Results We detected EGFR in 85 cases (90%), with overexpression in 33 cases (35%), and aberrant EGFR expression in the cytoplasm in 50 cases (53%). EGFR overexpression in the primary tumours was associated with lymph node progression, tumour–nodes– metastasis stage progression and proliferation (Ki-67 staining) in CSCC. EGFR overexpression and poor grade of differentiation were the strongest independent variables defining lymph node metastasis and progression in CSCC in a logistic regression model. Conclusions We demonstrate that EGFR overexpression has prognostic implications associated with lymph node metastasis and progression in CSCC.

None declared.

What’s already known about this topic?

J.P.-L. and C.R.-C. contributed equally to this article.

•

DOI 10.1111/bjd.14936

• •

Cutaneous squamous cell carcinoma (CSCC) is the second most frequent cancer in humans and sometimes exhibits a poor outcome. Epidermal growth factor receptor (EGFR) activation in keratinocytes induces cellular proliferation, increases cell survival and induces resistance to apoptosis. While the expression of EGFR has been extensively studied in CSCC, there is some controversy surrounding its prognostic significance.

What does this study add?

• © 2016 British Association of Dermatologists

Based on the results of our study, EGFR overexpression appears to have prognostic implications associated with lymph node metastasis and progression in CSCC.

British Journal of Dermatology (2017) 176, pp1279–1287

1279

~ueto et al. 1280 EGFR expression in cutaneous squamous cell carcinoma, J. Can

•

There was no association between the level of EGFR protein, detected by immunohistochemistry, and the level of EGFR RNA.

What is the translational message?

• •

We provide evidence that EGFR expression is a molecular marker for poor clinical outcome in cases of CSCC. EGFR-targeted therapies might be helpful in selected patients with metastatic CSCC.

Cutaneous squamous cell carcinoma (CSCC) is the second most frequent cancer in humans after basal cell carcinoma1–3 and its incidence is dramatically rising and is probably underestimated. More than 700 000 cases of CSCC are diagnosed in the U.S.A per year.4 The lifetime risk of developing CSCC has been estimated to be 7–11%.5 However, the incidence rate varies widely among geographical areas, with the highest incidence rates occurring in Australia and New Zealand.6 Given its high frequency, and although CSCC is rarely problematic, the absolute number of complicated cases is also high and CSCC is among the most costly forms of cancer.7 Thus, it is important to identify those cases of CSCC with a poor prognosis. Despite the significant progress made in the study of CSCC in recent years,8–11 many aspects concerning its prognosis still remain unknown. For that reason, the need to search for molecular markers that help to predict the biological behaviour of CSCC is imperative. Epidermal growth factor receptor (EGFR) ligands binding to EGFR lead to the activation of multiple downstream signalling pathways involved in cell proliferation, apoptosis, invasion, angiogenesis and metastasis.12,13 In squamous cells, EGFR plays an important role in regulating the RAS/MAPK, PI3K/AKT and phospholipase C pathways.14 These pathways are commonly altered in cancer, including in CSCC.15 EGFR activation in keratinocytes induces cellular proliferation with an increase in epidermal thickness and cellularity,16 and also increases cell survival and resistance to apoptosis.17 EGFR is necessary for maintaining the proliferative population in the basal compartment of epidermal tumours in animal models.18 While the expression of EGFR has been widely studied in CSCC, little is known about its prognostic significance. EGFR has been related to tumour progression in CSCC,19 but other published articles have failed to demonstrate this observation.20 EGFR-targeted therapies have emerged in recent years and, therefore, more indepth knowledge regarding the role of EGFR in CSCC is needed. Here, we analysed a series of 94 CSCCs and evaluated EGFR expression by immunohistochemistry and fluorescent in situ hybridization (FISH). We demonstrated that EGFR overexpression is associated with the progression and poor outcome of CSCC.

Patients and methods Clinical, epidemiological and pathological features in our series of cutaneous squamous cell carcinoma We evaluated a series of 94 CSCCs for which clinical, epidemiological and histopathological features are described in Table S1 (see Supporting Information). British Journal of Dermatology (2017) 176, pp1279–1287

We would like to clarify that immunosuppression was considered when (i) the patient was under treatment with systemic chemotherapy or with any immunosuppressive drug (including long-term corticosteroid use) or (ii) the patient had been diagnosed with a malignancy other than CSCC. Diabetes was not considered immunosuppressive.21 The location of the tumour was categorized as either (i) head and neck high risk, (ii) head and neck low risk or (iii) trunk and limbs. We followed the mask area to consider head and neck high-risk tumours; thus, head and neck high-risk locations were ear, lower lip, temple, nose, eyelid and the preauricular region. The grade of differentiation was classified as good, moderate or poor, according to previous reports.22 Desmoplasia was defined by the presence of thickening of collagen bundles around and inside the tumour, which involved at least 30% of the stroma.21 Tumours were staged according to the 7th edition of the American Joint Committee on Cancer Staging Guidelines (AJCC) for CSCC.23 Tissue microarray and immunohistochemistry Tissue samples embedded in paraffin and fixed in formalin were used to prepare tissue microarrays using a tissue arrayer device (Beecher Instruments, Silver Spring, MD, U.S.A). Three 1-mm diameter cylinders of each tumour were included to ensure quality, reproducibility and homogeneous staining of the slides. Three different areas within each tumour were selected to reproduce tumour heterogeneity, including tumour mass and the invasion front. For quantification of EGFR immunohistochemical staining we used anti-EGFR (HER-1, Dako, Glostrup, Denmark) prediluted by Dako (catalogue number K1492, EGFR pharmDxâ Kit for Dako Autostainer). A semiquantitative analysis of EGFR expression level was performed by three independent observers (J.C., C.R.-C. and E.C.). Staining intensity was classified as either weak (+), moderate (++) or strong (+++) (Fig. 1a). Protein overexpression was defined as those cases with strong (+++) staining intensity.19 These parameters were analysed in all three areas of each tumour collected. The intensity of EGFR expression was later transformed into a dichotomous variable with category 0 considered as nonintense expression in those cases with weak or moderate expression, and category 1 considered as overexpression in those cases with strong expression. We also considered the subcellular location of EGFR staining. We distinguished between aberrant expression, when present in the © 2016 British Association of Dermatologists


(a)

(b)

(c)

60

40

57

20 24

0

P = 0·007

Yes

Nodal progression

EGFR IH No overxpression Overexpression

40 56

20 24

9

4

No

60


Number of tumours

Number of tumours

P = 0·005

0

9

5

No

Yes

TNM progression

Fig 1. Epidermal growth factor receptor (EGFR) expression and cutaneous squamous cell carcinoma (CSCC) prognosis. (a) Examples of CSCC with different grade of EGFR expression by immunohistochemistry (IH); weak (+), moderate (++) and strong (+++). Upper figures original magnification 9 100 (round spots); lower figures original magnification 9 200 (rectangles). (b) EGFR overexpression in CSCC was associated with lymph node metastasis (v2-test) and (c) with the tumour–nodes–metastasis (TNM) stage progression (v2-test).

cytoplasm with or without membrane expression,24 or normal expression, present only in the plasmatic membrane. For quantification of Ki-67 immunohistochemical staining anti-Ki67 antibody (clone SP6, Master Diagnostica, Granada, Spain) was used at a 1 : 100 dilution. Ki-67 immunostaining was evaluated as the percentage of stained cells. An arithmetic median was calculated using the values from different © 2016 British Association of Dermatologists

cylinders in each tumour. Ki-67 expression was considered as a continuous variable. RNA extractions and reverse transcription RNA from tumours was extracted using QIAzolâ Lysis Reagent (Qiagen, Venlo, the Netherlands) and miRNeasy Mini Kit British Journal of Dermatology (2017) 176, pp1279–1287


(Qiagen). RNA quality was checked by spectrometer analysis (Nanodrop Products, Wilmington, DE, U.S.A.). Then 1 lg of total RNA was reverse transcribed using random hexamer primers with the Transcriptor First Strand cDNA Synthesis Kit (Roche, Pleasanton, CA, U.S.A.). Real-time quantitative polymerase chain reaction Real-time quantitative polymerase chain reaction was performed using a Mastercycler ep realplex4 S (Eppendorf, Stevenage, U.K.) and FAM-labelled TaqMan probes for EGFR (Hs01076090_m1, Thermo Fisher, Waltham, MA, U.S.A.) and GAPDH (Hs02758991_g1, Thermo Fisher). Amplifications were carried out using the PerfeCTaâ quantitative polymerase chain reaction SuperMix (Quanta Biosciences, Beverly, MA, U.S.A) in a reaction volume of 20 lL. Each reaction was performed in triplicate and the mean threshold cycle was calculated. GAPDH was used as the reference gene and the values obtained were related to a specific sample to calculate relative quantity of EGFR for each sample.

were more than 15 EGFR gene copies in 10% of the cells analysed or greater.25 Statistical analysis We performed statistical analyses with SPSS v.21 software (IBM, Armonk, NY, U.S.A.). We compared categorical variables with the v2-test or Fisher’s exact test when appropriate. The Kolmogorov–Smirnov test demonstrated that continuous variables did not follow a normal distribution. To compare two independent samples we used the Mann–Whitney U-test, and to compare more than two independent samples we used the Kruskal–Wallis test. For all tests P < 005 was considered to be significant. To build graphical representations of the statistical associations among variables, we used the Cytoscape (v.3.1.0) software freely available at www.cytoscape.org. We performed a logistic regression model to evaluate the independence of variables found to be significant and/or relevant in the univariate analyses.

Results Fluorescent in situ hybridization For FISH analysis 4-lm serial tissue sections containing representative tumour areas were sliced for each tumour. Cell copy numbers were evaluated by FISH using the LSI EGFR SpectrumOrange/CEP 7 SpectrumGreen probe (Vysis, Abbott Laboratories, Downers Grove, IL, U.S.A.). Paraffin-embedded tissue sections were deparaffined, dehydrated and air-dried. The slides were placed in 2 mmol L1 EDTA (pH 9) for 15 min. After cooling, the slides were transferred to a Coplin jar containing 40 mL of 09% NaCl (pH 15) and 160 mg of pepsin (Sigma-Aldrich Co., St Louis, MO, U.S.A.) preheated to 37 °C. Following incubation for 15 min, the sections were dehydrated in an alcohol series and fixed in 3 : 1 methanol : acetic acid solution for 10 min. Then, the slides were incubated in a hybridization chamber (Vysis) for 12 min at 72 °C, followed by 15–20 h at 37 °C. After hybridization, the slides were washed in standard saline citrate solutions. Nuclei were counterstained with 40 ,6-diamidino-2-phenylindole (Vector Laboratories Inc., Burlingame, CA, U.S.A.). The images were captured using an Olympus BX60 epifluorescence microscope (Olympus, Center Valley, PA, U.S.A.) coupled with a charge coupled device camera, and were then evaluated with the Cytovision software (Applied Imaging, Leica Biosystems, Newcastle upon Tyne, U.K.). Approximately 200 nonoverlapping tumour cells were evaluated. Samples were classified into the following two groups: (i) FISH negative, with no or low genomic gains (fewer than four copies of the gene in more than 40% of the cells) and (ii) FISH positive, defined as those samples with a high level of polysomy (more than four copies of the EGFR gene in at least 40% of the cells) or those samples with gene amplification. Gene amplification was considered when the ratio of copies per cell between the EGFR gene and the CEP7 chromosome probe was ≥ 2 or when there British Journal of Dermatology (2017) 176, pp1279–1287

Clinicopathological features of cutaneous squamous cell carcinoma were associated with lymphatic and tumour– nodes–metastasis progression In order to define the risk of progression of the CSCC more accurately, we evaluated the grade of association between the lymphatic and clinical stage of progression with the pathological features of the disease. Thus, we showed that lymph node metastasis was statistically associated with the presence of CSCC in high-risk locations of the head and neck (ear, lower lip, temple, nose, eyelid or preauricular region) (P = 0001), infiltrative growth pattern (P = 0012), poor grade of differentiation (P = 0003), perineural invasion (P = 0007) and greater tumour thickness (P = 0012). Also, lymph node metastasis was associated with the tumour stage at diagnosis, according to the tumour–nodes–metastasis (TNM) staging system of the AJCC (P = 0024) (Table 1). TNM progression was associated with the same clinical and pathological variables as lymph node metastasis including tumour locations of high risk (P = 0001), infiltrative growth pattern (P = 0009), poor grade of differentiation (P = 0001), perineural invasion (P = 0004) and greater tumour thickness (P = 0027). In addition, TNM progression was associated with the tumour stage at diagnosis, according to the TNM staging classification of the AJCC (P = 0016) (Table 1). Thus, we identified several clinical and pathological characteristics of CSCC associated with poor clinical evolution of the disease. Although immunosuppression is a well-known feature of poor prognosis in CSCC, we did not find it to be associated with poor outcome in CSCC. In our study, we did not find an association between poor grade of differentiation or other histopathological tumour traits of poor outcome and immunosuppression. Also, there was no association between immunosuppression and events of poor clinical evolution, local © 2016 British Association of Dermatologists

~ueto et al. 1283 EGFR expression in cutaneous squamous cell carcinoma, J. Can Table 1 Associations of several histopathological tumour features and epidermal growth factor receptor (EGFR) expression with lymph node progression and tumour–nodes–metastasis (TNM) stage of progression Histopathological tumour feature

Yes

Lymphatic progression Growth pattern Infiltrative 11 Noninfiltrative 2 Grade of differentiation Poor 7 Good/moderate 6 Lymphovascular invasion Yes 4 No 8 Perineural invasion Yes 7 No 6 Location HN high risk 12 HN low risk 1 Trunk and extremities 0 Thickness, mm 8 (65) (Median/IQR) EGFR Overexpression 9 Nonoverexpression 4 TNM stage of progression Histopathological tumour feature Stage at diagnosis I 1 II 13 Growth pattern Infiltrative 12 Noninfiltrative 2 Grade of differentiation Poor 8 Good/moderate 6 Lymphovascular invasion Yes 4 No 77 Perineural invasion Yes 8 No 6 Location HN high risk 13 HN low risk 1 Trunk and extremities 0 Thickness, mm 725 (525) (Median/IQR) EGFR Overexpression 9 Nonoverexpression 5

No

P-value

39 44

0012

12 71

0003

2 72

0002

14 69

0007

32 37 14 575 (43)

0001

0012a

24 57

0005

34 50

0016

40 44

0009

11 73

0001

3 10

0009

15 69

0004

33 34 14 6 (44)

0001

24 56

0007

027

HN, head and neck; IQR, interquartile range. aMann–Whitney Utest.

recurrence and nodal progression. We attribute the lack of influence of immunosuppression on prognosis in our cohort to the small number of patients with immunosuppression included in the study (n = 10). © 2016 British Association of Dermatologists

Epidermal growth factor receptor expression in the primary tumour correlated with poor clinical outcome The use of EGFR expression to predict CSCC prognosis is controversial.19,20 To address this, we determined the EGFR expression in primary tumours by immunohistochemistry (Fig. 1a). We found that EGFR was expressed in 85 of the 94 cases (90%) surveyed; of these, 52 cases (55%) showed moderate expression and 33 cases (35%) showed overexpression. The remaining nine cases were negative for EGFR staining. Interestingly, we found that the overexpression of EGFR in the primary tumour was associated with the development of lymph node metastases (P = 0005), and with the progression of the TNM stage during follow-up (P = 0007) (Fig. 1b, c and Table 1). We did not find an association between EGFR expression and local recurrence, or any other clinical and pathological characteristics of CSCC. In addition, EGFR overexpression was associated with a higher proliferative index measured by Ki-67 expression. Thus, Ki-67 was higher in tumours with intense EGFR expression (P = 0025) (Fig. 2a–c). Later, we evaluated the location of EGFR expression in CSCC cells. EGFR was mostly located in the plasmatic membranes as expected, but we also found an aberrant pattern of EGFR expression in the cytoplasm24 in 50 cases (53%) (Fig. 3a, b). Moreover, we found a statistical association between the aberrant pattern of EGFR expression and the moderate/poor expression compared with the good grade of differentiation (P = 0004) (Fig. 3c). Overexpression of epidermal growth factor receptor was associated with gains of the EGFR gene in a small proportion of cutaneous squamous cell carcinoma cases To investigate the mechanism of overexpression of EGFR in cases of CSCC we evaluated the amplification of the EGFR gene. EGFR amplifications were detected by FISH in only six of 84 tumours (7%) that were positive for EGFR expression by immunohistochemistry (one case was not available for FISH analysis). We did not identify changes in the EGFR locus in the remaining tumours. In our study, EGFR amplification was significantly associated with EGFR overexpression (P = 0028) (Fig. 2d–f), but we did not identify an association between the FISH results and the clinical or pathological features of the disease, perhaps because of the few cases of EGFR amplification detected. Subsequently, we assessed whether overexpression of the EGFR protein could be due to increased gene expression. Hence, we evaluated the expression of EGFR in 32 randomly selected cases. We found no correlation between EGFR overexpression and transcript levels (Fig. 3d). Epidermal growth factor receptor overexpression is an independent prognostic factor in cutaneous squamous cell carcinoma To predict the risk of developing lymph node metastases and the risk of TNM progression in CSCC, we generated British Journal of Dermatology (2017) 176, pp1279–1287


(a) (c)

(b)

% Ki-67 expression

50·00

P = 0·025

40·00

30·00

20·00

10·00

0·00

–10·00

Overexpression

No overexpression

EGFR (IH)

(d)

(e)

Number of tumours

(f)

60

P = 0·028 50


40

30 51

20 27

10 1

5

0

No changes

Amplification

EGFR FISH

Fig 2. Epidermal growth factor receptor (EGFR) expression, tumour proliferation and EGFR amplification. (a, b) Analysis of cutaneous squamous cell carcinoma (CSCC) proliferation by Ki-67 expression determined by immunohistochemistry (IH). (a) A case of CSCC with low Ki-67 expression. Left, original magnification 9 100 (round spot). Right, original magnification 9 200 (square). (b) The figure shows a case of CSCC with high Ki-67 expression. The distribution of the panels is the same as in (a). (c) Comparison of proliferation (Ki-67 expression) in tumours with and without EGFR overexpression (Mann–Whitney U-test). (d, e) Analysis of EGFR gene amplification by fluorescent in situ hybridization (FISH). (d) A case of CSCC cells without gene amplification. (e) A case of CSCC with EFGR gene amplification. (f) Distribution of tumours with EGFR expression or overexpression by immunohistochemistry in groups with or without EGFR gene amplifications detected by fluorescent in situ hybridization (FISH) (v2-test).

logistic regression models. As predictor variables, we used clinical and pathological characteristics of the CSCCs including location, tumour thickness, tumour size, growth pattern, British Journal of Dermatology (2017) 176, pp1279–1287

grade of differentiation, perineural invasion and grade of proliferation, together with EGFR expression. The logistic regression models showed that lymph node progression was © 2016 British Association of Dermatologists


(a)

(c) P = 0·004

30 41

20

1

n = 13

24 9

0

EG

Aberrant pattern of EGFR expression

no ti nt en se

Yes

FR

No

n = 19

in te ns e

20

0

2

FR

10

n.s

3

EGRF RQ

40

(d)

Good Moderate / poor

EG

50

Number of tumours

Fig 3. Aberrant pattern of epidermal growth factor receptor (EGFR) expression in cutaneous squamous cell carcinoma (CSCC). (a) A case of CSCC with a normal pattern of EGFR expression in the plasmatic membrane detected by immunohistochemistry. The upper figures show staining; original magnification 9 100 (round spot) and original magnification 9 200 (rectangle). (b) A case of CSCC with aberrant overexpression of EGFR in the cytoplasm. The distribution of the panel is the same as in (a). (c) Distribution of the aberrant pattern of EGFR expression in different groups of CSCC based on the grade of differentiation (v2-test). (d) Comparison of EGFR mRNA levels in tumours with high and low protein expression by immunohistochemistry (t-test). NS, nonsignificant. RQ: relative quantity in realtime polymerase chain reaction.

(b)

Table 2 Prediction of nodal progression and tumour–nodes–metastasis (TNM) stage of progression by logistic regression in cutaneous squamous cell carcinoma Logistic regression model

Independent factors

Odds ratio

Confidence interval

P-value

Nodal metastases

EGFR overexpression Grade of differentiation Grade of differentiation EGFR overexpression Perineural invasion

7117 5664 6557 4858 3324

185–2737 145–2211 166–2591 123–1923 0873–12652

0004 0013 0007 0024 0078

TNM stage progression

EGFR, epidermal growth factor receptor.

associated with the poor grade of differentiation (P = 0004) and with intense EGFR expression (P = 0013); in addition, the progression of the TNM was associated © 2016 British Association of Dermatologists

with the same variables in an analogue multivariate model, i.e. the grade of differentiation (P = 0007) and EGFR overexpression (P = 0024) (Table 2). Thus, we confirmed the



importance of EGFR expression in primary CSCC to predict prognosis.

Discussion There is a lack of knowledge concerning molecular markers with potential prognostic value for CSCC. Here, we demonstrated that EGFR overexpression in the primary tumour was associated with lymphatic progression, the TNM stage of progression in CSCC and with tumour proliferation (Figs 1, 2c and Table 1). We also demonstrated that both EGFR overexpression and the grade of differentiation were independent prognostic factors for node metastasis and progression of TNM in CSCC in multivariate analysis (Table 2). To achieve this, we retrospectively selected 94 cases of CSCC, some of them with poor prognosis, in order to have a larger sample size representing this outcome in the study cohort. We used a semiquantitative method for the evaluation of EGFR expression by immunohistochemistry. Visual analysis of immunohistochemistry has a subjective component,26 but it has been shown to have a high correlation with quantification using new imaging techniques,27,28 thus indicating that this semiquantitative visual evaluation system is useful despite its limitations. There is little information and some controversy concerning the prognostic implications of EGFR overexpression in CSCC.24 Indeed, EGFR overexpression seems to facilitate the acquisition of a more aggressive phenotype of CSCC.29,30 Although EGFR was found to be overexpressed in CSCCs with no association with prognosis,20,31 other studies have shown that EGFR overexpression was more common in primary tumours of the head and neck that progressed.19 Here, we found that EGFR overexpression is associated with poor outcome in CSCC (Tables 1, 2). It has been proposed that EGFR aberrant expression in the cytoplasm is involved in signalling pathways responsible for cell proliferation, survival, invasion, angiogenesis and metastasis in many types of cancers, including head and neck squamous cell carcinoma (SCC).32–34 The loss of the membrane signal of EGFR and its accumulation in the cytoplasm has also been observed in CSCC, and it has been suggested that this is a result of EGFR mutations.35 In this study, we found a statistical association between the aberrant pattern of EGFR expression and the moderate/poor grade of differentiation (Fig. 3c). In addition, EGFR expression has been associated with proliferation,24,36 which is consistent with the association between EGFR expression and Ki-67 found in this study (Fig. 2c and Table 1). This suggests that the EGFR signalling pathway may be implicated in the proliferation of CSCC. We observed that EGFR overexpression was much more common than gains of the EGFR locus, suggesting that the overexpression of EGFR in CSCC does not directly depend on genomic changes (Fig. 2a, b, e). This low frequency of EGFR gene amplification has also been reported in head and neck cell carcinoma.37,38 Other authors have also evaluated EGFR copy number aberrations using FISH in CSCC, with British Journal of Dermatology (2017) 176, pp1279–1287

controversial results concerning its association with EGFR expression and its relevance in prognosis.19,31 Similarly, we did not find a correlation between EGFR protein overexpression and RNA levels (Fig. 3d). The absence of an association between the level of EGFR protein, detected by immunohistochemistry, and the level of EGFR RNA is not uncommon, and has also been described for head and neck SCC37 and nonsmall cell lung cancer.32 In relation to this, it has been suggested that the overexpression of EGFR in head and neck SCC could be due to its reduced degradation and dephosphorylation; this might also be the case in CSCC.37 In conclusion, we have demonstrated that EGFR overexpression has prognostic implications, and is associated with lymph node metastasis and progression. Thus, we provide evidence that EGFR expression is a molecular marker for poor clinical outcome in cases of CSCC. In addition, EGFR-targeted therapies might be helpful in selected patients with metastatic CSCC, although additional studies need to be carried out to establish this possibility.

Acknowledgments J.P.L. was partially supported by FEDER and MICINN (PLE2009-119, SAF2014-56989-R), Instituto de Salud Carlos III (PI07/0057, PI10/00328, PIE14/00066), Junta de Castilla y Le on (SAN673/SA26/08, SAN126/SA66/09, SA078A09, CSI034U13, BIO/SA31/15), IBSAL (IBY15/00003), the ‘Eugenio Rodrıguez Pascual’, the ‘Fundaci on Inbiomed’ (Instituto Oncol ogico Obra Social de la Caja Guipozcoa-San Sebastian, Kutxa), and the ‘Fundaci on Sandra Ibarra de Solidaridad frente al Cancer’. C.R.-C. is funded by Q3718001E (2009-2010) and GRS 612/A/11 (2011-2012) and ‘the Fundaci on Eugenio Rodrıguez Pascual’. A.C.-M. was supported by FIS (PI07/0057) and MICINN (PLE2009-119). We thank Emma Keck for English editing and Angustias Perez Sanchez for technical assistance.

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Supporting Information Additional Supporting Information may be found in the online version of this article at the publisher’s website: Table S1. Clinicoepidemiological and histopathological tumour features evaluated in the cutaneous squamous cell carcinoma study cohort.
