ANTICANCER RESEARCH 38 : 795-802 (2018) doi:10.21873/anticanres.12286

PDGFR α/ HER2 and PDGFR α/ p53 Co-expression in Oral Squamous Cell Carcinoma PIOTR CIERPIKOWSKI 1, ANNA LIS-NAWARA 1, PAWEL GAJDZIS 2 and JULIA BAR 1

Department of 1Immunopathology and Molecular Biology, and 2Pathomorphology and Oncological Cytology, Wroclaw Medical University, Wroclaw, Poland

Abstract. Aim: The purpose of this study was to explore the heterogeneous histology, tumor site and biological diversity of parallel expression of platelet-derived growth factor α oral tumors (3, 5). The pathomorphological features of oral (PDGFR α) and human epidermal 2 carcinomas are often associated with overexpression/ (HER2) or p53 in relation to clinicopathological parameters of amplification of surface receptors and / oral squamous cell carcinoma (OSCC) to define their role in overexpression suppressor in tumor cells (4, 6). progressive growth of tumor. Materials and Methods: Expression Therefore, an examination of the biological features of oral of PDGFR α, HER2 and p53 was evaluated in 71 OSCC samples carcinomas with established clinical and pathomorphological by immunohistochemistry. HER2 status was verified by parameters might be useful for screening subgroups of patients fluorescence in situ hybridization. Results: PDGFR α and p53 for individual therapy (7). Currently, among the biological expression were associated with tumor grade (p=0.043 and factors, receptors [ e.g. platelet-derived growth p=0.040, respectively). HER2 expression was more frequent in factor receptors (PDGFRs), human advanced (III/IV) cancer (p=0.006). A positive correlation of receptor 2 (HER2)] and p53 are being intensively PDGFR α with HER2 (r=0.267; p=0.024) and with p53 investigated in solid tumors including OSCC (4, 5). (r=0.266; p=0.025) was noted. PDGFR α/ HER2 and PDGFR α is a member of the transmembrane receptor PDGFR α/ p53 co-expression was found more often in G3 than tyrosine kinases family which plays a key role in the in G1 and G2 tumors (p=0.008 and p=0.015, respectively). regulation of cell proliferation, chemotaxis and Conclusion: Our study revealed that PDGFR α/ HER2 and tumorigenesis by autocrine and paracrine stimulation (8, 9). PDGFR α/ p53 co-expression exists in poorly differentiated PDGF is involved in angiogenesis in normal and tumor OSCCs, suggesting that cooperation between these tissue (10). The dysfunction of PDGFs and their cognate might enhance aggressive behavior of tumor. receptors has been shown to play an important role in human carcinogenesis (11). Overexpression of PDGFR was Oral squamous cell carcinoma (OSCC) is among the 10 most associated with high malignancy of gliomas and advanced common malignancies in the world and its incidence has stage of head and neck carcinomas (10, 11). PGDFRs have escalated globally (1, 2). Despite recent advances in been very rarely studied in OSCC and their role has not been available therapeutic strategies, a high percentage of patients established (9). still have a poor response to therapy and high recurrence The second receptor belonging to the family of tyrosine rates (2). The 5-year survival rate for OSCC patients has not receptors is HER2 (ERBB2). HER2 is a member of the improved and remains in the range of 50-60% (3). epidermal growth factor receptor (EGFR) family involved in The molecular mechanisms responsible for the development controlling cell growth and differentiation (5). In normal and progression of oral cancer are still poorly understood (3, cells, the expression and activity of HER2 is strictly 4). Genetic and molecular studies are limited by the controlled (5). HER2 overexpression has been observed in many cancer types such as breast, gastric, colon, head and neck carcinoma, which are often associated with increased tumor size, tumor grade, metastasis activity and hence a poor Correspondence to: Piotr Cierpikowski, Department of prognosis (7, 12, 13). Immunopathology and Molecular Biology, Wroclaw Medical The tumor suppressor TP53 plays critical role in University, Borowska 213, 50-556 Wroclaw, Poland. Tel: +48 717343960, Fax: +48 717343968, e-mail: [email protected] repressing cancer invasion and metastatic progression. Recent evidence indicates that alteration in the TP53 gene represents Key Words: PDGFR α, HER2, p53, oral squamous cell carcinoma, common molecular changes in human cancer associated with immunohistochemistry, FISH technique. a selective growth advantage and loss off of cell-cycle control

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(4, 14). The loss of p53 function with resultant overexpression Table I. Clinicopathological characteristics of patients with oral has been reported in various human tumors such a breast, squamous cell carcinoma. brain, rectum, colon, oesophagus, lung cancers and OSCCs Parameter N (%) (4). Previous studies have shown that oral carcinomas with overexpression/mutation of p53 grow faster and have Age aggressive nature and poor prognosis (1, 3, 15). <62 Years 31 (43.7) Up to now, co-expression of PDGFR α/ HER2 and ≥62 Years 40 (56.3) Gender PDGFR α/ p53 in oral carcinoma has not been examined and Female 23 (32.4) the relationship between these proteins which play different Male 48 (67.6) functions in tumor cells during tumor growth remains Tumor grade unclear. It seems to be important to analyze correlations 1 27 (38.0) between PDGFR α, which promotes angiogenesis of tumor, 2 36 (50.7) 3 8 (11.3) in relation with HER2 and p53 expression in OSCC. Tumor site The aim of this study was to evaluate parallel expression Tongue 30 (42.3) of PDGFR α and HER2 and p53 protein in OSCC in relation Flour of the mouth 18 (25.4) to clinicopathological parameters in order to define whether Oral vestibule 19 (26.8) co-expression of these proteins determines the individual Hard palate 4 (5.6) T Stage clinicopathological features of OSCCs and may be useful T1 11 (15.5) biomarkers characterizing their growth and behavior. T2 20 (28.2) T3 26 (36.6) Materials and Methods T4 14 (19.7) Lymph node metastasis Patient tissues. A total 71 cases of excisional surgical specimens from Yes 29 (40.8) 71 patients treated surgically for primary OSCC were obtained from No 42 (59.2) the Department of Pathomorphology and Oncological Cytology of the Clinical stage Wroclaw Medical University, Poland between 2011-2014. Only the I/II 25 (35.2) following tumor sites were included in the study: tongue, floor of the III/IV 46 (64.8) mouth, oral vestibule and hard palate. Patients with other localization of tumor, non-OSCC histopathology or treated before surgery were excluded. All tumors were histologically verified to confirm the diagnosis and categorize tumor grade, as well-, moderately or poorly differentiated according to the WHO classification (16). Clinical data with primary antibodies (anti-p53, dilution 1:50; anti-HER2, for all 71 patients were analyzed to identify age, gender, tumor site dilution 1:80; anti-PDGFR α, dilution 1:40) overnight at 4˚C. After and clinical stage. Clinical stage was classified in accordance with washing with 0.1 M Tris-buffer, pH 7.4 (TBS), the tissue samples the 2009 American Joint Committee on Cancer staging criteria were incubated (15 minutes at room temperature) with a secondary (seventh edition) (17). The distribution of clinical and histological biotinylated antibody and with streptavidin-horseradish peroxidase- features of the patients is shown in Table I. This study was approved conjugated (EnVision detection Kit Peroxidase, DAB rabbit/mouse; by the Ethics Committee of Wroclaw Medical University (approval Dako,). Following washing with TBS, the immunohistochemical date 18th May 2016, no. KB-230/2016) and was performed in reaction was detected by 3,3’-diaminobenzidine (Dako) as a accordance with the declaration of Helsinki. chromogen (8 minutes at room temperature). Slides were then counterstained with hematoxylin. Internal positive controls were set Immunohistochemistry (IHC). Immunohistochemical staining of as: astrocytoma for PDGFR α, breast carcinoma for HER2, colon PDGFR α, HER2, and p53 was performed on paraffin-embedded carcinoma for p53. Negative controls were performed without tissues from the selected blocks using the ABC method consisting primary antibody. of labeled streptavidin biotin reagents conjugated to peroxidase (LSAB+ Kit, HRP; Dako, Copenhagen, Denmark) and the following Interpretation of IHC reaction. The slides were evaluated under an primary monoclonal antibodies: anti-PDGFR α ( clone D13C6; Cell Olympus BX-51 double-headed microscope (Olympus, Tokyo, Signaling Technology, Danvers, MA, USA ), anti-HER2 (clone Japan). The localization, distribution and intensity of PDGFR α, CB11; Novocastra, Newcastle, United Kingdom) and anti-p53 HER2 and p53 immunopositivity were evaluated in the tissue (clone DO-7; Novocastra). All procedures were performed sections. Expression of PDGFR α and HER2 was assessed by according to the manufacturer’s protocols. determination of membranous immunostaining based on the Five-micrometer sections from each selected block were intensity of immunostaining and the percentage of stained tumor deparaffinized and antigen-retrieved in citrate buffer (pH=6.0) by tissue area. Immunostaining for PDGFR α and HER2 exceeding microwaving at 700W for three times for 5 minutes for each more than 10% of tumor cells was considered as positive. The antibody. After microwave heating, the samples were cooled for 20 percentage of p53-positive cells was determined by counting 1000 minutes. The activity of endogenous peroxidase was blocked by cells in 10 randomly selected high-power fields in relation to the using 3% H 2O2. Nonspecific binding of antibodies was blocked total number of cells. Immunoreactivity was judged positive if with 10% bovine serum albumin. Tissue preparations were treated immunostaining of p53 protein was observed in more than 10% of

796 Cierpikowski et al : PDGFR α/ HER2 and PDGFR α/ p53 Co-expression in Oral Carcinoma

Table II. Association between clinicopathological parameters and protein expression in patients with oral squamous cell carcinoma.

PDGFR α, n (%) HER2, n (%) p53, n (%)

Parameter Negative Positive p-Value Negative Positive p- Value Negative Positive p-Value

Age <62 Years 19 (61.3) 12 (38.7) 0.748 a 7 (22.6) 24 (77.4) 0.994 a 21 (67.7) 10 (32.3) 0.034 a ≥62 Years 26 (65.0) 14 (35.0) 9 (22.5) 31 (77.5) 17 (42.5) 23 (57.5) Gender Female 13 (56.5) 10 (43.5) 0.406 a 6 (26.1) 17 (73.9) 0.620 a 15 (65.2) 8 (34.8) 0.171 a Male 32 (66.7) 16 (33.3) 10 (20.8) 38 (79.2) 23 (47.9) 25 (52.1) Tumor grade 1 17 (63.0) 10 (37.0) 5 (18.5) 22 (81.5) 17 (63.0) 10 (37.0) 2 26 (72.2) 10 (27.8) 0.043 a 11 (30.6) 25 (69.4) 0.142 a 20 (55.6) 16 (44.4) 0.040 a 3 2 (25.0) 6 (75.0) 0 (0.0) 8 (100.0) 1 (12.5) 7 (87.5) Tumor site Tongue 19 (63.3) 11 (36.7) 7 (23.3) 23 (76.7) 13 (43.3) 17 (56.7) Flour of the mouth 13 (72.2) 5 (27.8) 0.618 a 4 (22.2) 14 (77.8) 0.997 a 11 (61.1) 7 (38.9) 0.494 a Oral vestibule 10 (52.6) 9 (47.4) 4 (21.1) 15 (78.9) 12 (63.2) 7 (36.8) Hard palate 3 (75.0) 1 (25.0) 1 (25.0) 3 (75.0) 2 (50.0) 2 (50.0) T Stage T1 8 (72.7) 3 (27.3) 2 (18.2) 9 (81.8) 7 (63.6) 4 (36.4) T2 13 (65.0) 7 (35.0) 0.849 a 6 (30.0) 14 (70.0) 0.818 a 12 (60.0) 8 (40.0) 0.669 a T3 15 (57.7) 11 (42.3) 5 (19.2) 21 (80.8) 13 (50.0) 13 (50.0) T4 9 (64.3) 5 (35.7) 3 (21.4) 11 (78.6) 6 (42.9) 8 (57.1) Lymph node metastasis Yes 21 (72.4) 8 (27.6) 0.189 a 11 (26.2) 31 (73.8) 0.375 a 21 (50.0) 21 (50.0) 0.474 a No 24 (57.1) 18 (42.9) 5 (17.2) 24 (82.8) 17 (58.6) 12 (41.4) Clinical stage I/II 17 (68.0) 8 (32.0) 0.551 a 8 (32.0) 17 (68.0) 0.006 b 15 (60.0) 10 (40.0) 0.420 a III/IV 28 (60.9) 18 (39.1) 8 (17.4) 38 (82.6) 23 (50.0) 23 (50.0)

PDGFR α: Platelet-derived growth factor receptor α; HER2: human epidermal growth factor receptor 2. aChi-square test. bMann-Whitney U-test. Bold values indicate statistical significance ( p< 0.05).

tumor cells. The intensity of immunoreaction was scored as: rinsed in purified water for 5 minutes and washed twice in standard negative, weak, moderate, and strong. The immunostained slides saline citrate (SSC) for 5 minutes. The slides were then heated at were independently evaluated by two of the Authors who were 80˚C in sodium thiocyanate solution for 30 minutes, followed by blinded to clinical information of individual patients. The rinsing for 1 minute in distilled water and washing in 2×SSC. The percentage of PDGFR α-, HER2- and p53-positive tumor cells were sections were then subjected to protease at 37˚C for 35 minutes, divided into four groups according to immunoreactivity and staining washed in 2×SSC for 5 minutes and air-dried. After that HER2 probe intensity as follows: negative: 0-10% positively stained cells, added to each slide which was then coverslipped. Samples were weakly positive: 11-30% positively stained cells, moderately denatured for 5 minutes at 72˚C. After that hybridization was positive: 31-50% positively stained tumor cells, strongly positive: performed for at least 16 hours at 37˚C in DAKO Hybridizer™ more than 50% positively stained cells. equipment. Then preparations were washed with 2×SSC at room temperature. After coverslips were removed, slides were immersed Fluorescence in situ hybridization (FISH). FISH technique was in 2×SSC/0.3% NP40 at 72˚C. After air-drying in the dark, slides performed on thin formalin-fixed paraffin-embedded tissue sections were counterstained with 4’6’-diamino-2-phenylindole (DAPI). The (n=17 cases with weak, moderate and strong HER2 slides were examined using an Olympus BX-61 fluorescence immunoreactivity) using -specific identifier Pathvysion HER-2 microscope (Olympus, Tokyo, Japan) with filters suitable for Probe Kit (Vysis, Downers Grove, IL, USA). Spectrum Orange fluorescein/rhodamine and DAPI. Images were analyzed using CellF HER2 probe located at 17p11.2-q12 was used together Imaging Software (Olympus, Tokyo, Japan). The methodology was with Spectrum Green Dual Colour probe located at the centromere performed according to ISO/IEC 17025:2005 accreditation. HER2 of chromosome 17 (17p11.1-q11.1) according to the methodological gene amplification was considered as positive when the FISH ratio procedure of Abbott/Vysis. Sections were deparaffinized in xylene was higher than 2.2 or HER2 gene copy was more than 6.0. In cases (3×5 minutes) and dehydrated in 99.8% ethanol (3×1 minute). Tissue with polymorphism of HER2 gene, polysomy of chromosome 17 was samples were pre-treated by immersing in 0.2 N hydrochloric acid excluded by TP53 gene analysis using Vysis LSI TP53 (17p13.1) for 20 minutes to avoid tissue autofluorescence, then the slides were Spectrum Orange Probe (Vysis, Downers Grove, IL, USA) (18).

797 ANTICANCER RESEARCH 38 : 795-802 (2018)

Figure 2. Representative case of oral squamous cell carcinoma analyzed for human epidermal growth factor receptor 2 (HER2) gene status by fluorescence in situ hybridization technique. Positive (A) and negative (B) for HER2 gene polymorphism in oral squamous cell carcinoma, Green: Centromere region; orange: HER2 gene. Magnification ×600.

p< 0.05. Statistical tests were performed using STATISTICA v12.0 (StatSoft, Krakow, Poland).

Results

The presence of PDGFR α was observed on the membrane of Figure 1. Membranous expression of platelet-derived growth factor tumor cells in 26/71 (36.6%) cases. Positivity for PDGFR α receptor α ( PDGFR α) ( A) and human epidermal growth factor receptor was found at various levels in tumor tissue (11-70%), but most 2 (HER2) (B) in cancer cells. Strong nuclear expression of p53 protein positive cases (80.8%) presented weak intensity and in cancer cells (C) (EnVision technique). Scale bar=50 μm. immunoreactivity (Figure 1A). In some cases of OSCC, PDGFR α expression was observed in both tumor and stromal cells. PDGFR α expression was seen more frequently in grade (G) 3 tumors compared to G1 and G2 tumors ( p= 0.043). Statistical analysis. Correlations between PDGFR α, HER2, p53 expression and clinicopathological parameters were statistically There were no statistical correlations noted between PDGFR α studied by chi-square test or nonparametric tests. Associations expression and other clinicopathological parameters (Table II). between proteins were analyzed by Spearman’s correlation. Membranous HER2 immunostaining was noted in 55 out Differences were considered as statistically significant when of 71 (77.5%) cases and ranged from 11 to 90% of tumor

798 Cierpikowski et al : PDGFR α/ HER2 and PDGFR α/ p53 Co-expression in Oral Carcinoma

Table III. Spearman’s rank correlation between expression levels of studied proteins in oral squamous cell carcinomas.

PDGFR α HER2 p53

r p-Value r p- Value r p-Value

PDGFR α 0.267 0.024 0.266 0.025 HER2 0.267 0.024 0.093 0.440 p53 0.266 0.025 0.093 0.440

PDGFR α: Platelet-derived growth factor receptor α; HER2: human epidermal growth factor receptor 2. Bold values indicate statistical significance ( p< 0.05).

tissue (Figure 1B). Most OSCCs showed strong intensity and high immunoreactivity for HER2 in more than 50% of tumor tissue (43.6% of all positive cases). It was revealed that HER2 expression was not comparable with HER2 gene status: HER2 gene polymorphism was present in 2/17 (11.8%) cases (Figure 2). We found that HER2 immunoreactivity was more frequent in advanced (III/IV) clinical stage of disease as compared with low (I/II) stage ( p= 0.006). No correlations between HER2 expression and other clinicopathological parameters were observed (Table II). Expression of p53 was found in 33/71 (46.5%) tumors. Nuclear accumulation was observed in different ranges from 11 to 90% tumor tissue (Figure 1C). Strong intensity and Figure 3. Correlation between protein immunophenotypes and immunoreactivity was observed in 33.3% of all positive clinicopathological parameters of oral squamous cell carcinomas. High cases and dominated in glandular structure of tissue. No tumor grade was associated with platelet-derived growth factor receptor α/ human epidermal growth factor receptor 2 (PDGFR α+/HER2 +) positive reaction was observed in stroma cells. P53 + + expression was associated with patient age ( p= 0.034). The immunophenotype (p=0.008) (A), and with PDGFR α /p53 immunophenotype (p=0.015) (B). presence of p53 was significantly more often in poorly differentiated carcinomas (G3) than in moderately (G2) and well-differentiated tumors (G1) ( p= 0.040). There were no statistical correlations between p53 expression and other clinicopathological features (Table II). Discussion Interestingly, we found positive correlation of expression of PDGFR α with HER2 (r=0.267, p= 0.024) and with p53 In the present study, we analyzed co-expression of PDGFR α (r=0.266, p= 0.025) expression by IHC in the group of OSCCs with HER2 or p53 in OSCC samples to investigate the role analyzed ( Table III ). To determine the association between co- of these molecules in progressive growth of tumor and expression of PDGFR α/ HER2 and PDGFR α/ p53 in relation understanding of mechanisms responsible for the aggressive to clinicopathological parameters of OSCCs, the total group nature of OSCC. To our best knowledge, this is the first of oral carcinomas was subdivided into two subgroups report describing co-expression of PDGFR α with HER2 or according to co-expression-positive and co-expression- p53 in relation to clinicopathological parameters of OSCC. negative cases. Further analysis between co-expression of PDGFR α immunohistochemical expression in OSCC has studied biomarkers and clinicopathological parameters of been analyzed very rarely and its role in tumor growth is still OSCCs was performed separately by subgroup and compared. controversial. Several authors indicated that autocrine and As shown in Figure 3, the co-expression of PDGFR α/ HER2 paracrine stimulation of PDGFR α could play a crucial role in and PDGFR α/ p53 was found more often in poorly (G3) than oral and head and neck tumorigenesis and progression (8-10). in well- (G1) and moderately (G2) differentiated tumors; the One study showed that during tumor growth, PDGFR α observed differences were statistically significant ( p= 0.008 stimulation can initiate the activation of different signaling and p= 0.015, respectively). receptors, e.g. EGFR and Notch, which leads to dedifferentiation

799 ANTICANCER RESEARCH 38 : 795-802 (2018) of tumor cells, and increased cell motility and aggressiveness of accumulation of p53 protein in an inactive form might play tumor (11). In the present study, PDGFR α expression observed a key role in tumorigenesis and progression of OSCC (3). in some sets of oral carcinomas might increase activation of Therefore, it seems reasonable to assume that high p53 signaling pathways in tumor cells which depend on this receptor protein expression levels detected by IHC reflect the (8). We might suggest that oral carcinoma cells with higher extended half-life of p53 protein encoded by TP53 gene PDGFR α expression might present more aggressive behavior mutation (3). Similarly to published studies (3, 14, 15) we (11). Our suggestion is supported by the association revealed found correlation between p53 overexpression and grade of between PDGFR α expression and high grade of OSCC found in OSCC. The authors postulated that patients with OSCC the current study which may be partly comparable to other whose tumor showed high expression of p53 protein had results which suggest that this receptor is a crucial factor in the worse clinicopathological parameters and presented regulation of angiogenesis (8, 11). Some authors postulated that aggressive behavior (3, 14). In contrary to earlier data (26), PDGFR α might indirectly facilitate spread of carcinoma cells we found p53 expression more frequently in tumor of from primary tumor tissue and invasion of surrounding normal patients over 62 years old than those younger. Results from tissue (8, 9). In our opinion, the association between PDGFR α the current study indicate that in older patients with OSCC, expression and poorly differentiated tumor cells might have the risk of p53 alteration is higher and may be the result of influence cell motility and facilitate their migration through the accumulation of p53 DNA damage. No significant basement membrane. Similarly to liver and breast carcinoma, differences between p53 expression according to clinical high PDGFR α expression in OSSC might contribute to reduced tumor stage were observed in the present study, indicating cell–cell adhesion and increased spread of carcinoma cells (19). that p53 alteration might be early event in oral carcinoma In vitro experimental study revealed that suppression of growth (4, 26, 27). However, there are data (3, 14) of p53 PDGFR α/β tyrosine phosphorylation inhibits carcinoma cell protein expression being more frequent in advanced clinical migration (10). This confirms the essential role of PDGFR α in stage of tumor. tumor cell invasion (10). The current study is the first that revealed the co- Early reports of HER2 overexpression in head and neck expression of PDGFR α with HER2 as well as with p53 squamous cell carcinomas range from 0-47% (20). However, protein in oral carcinomas. There are no data as far as we are in laryngeal cancer, positive HER2 immunostaining was aware analyzing the correlation between these proteins in found in 68.0% of cases (21). In our study, IHC staining of oral cancer but results of this study might be partly HER2 was observed in 77.5% of OSCC. These results are comparable with other studies (3, 14, 26, 27) that found similar to those of Dalal et al. (13), who detected HER2 positive correlation between p53 and other proteins such as positivity in 62.2% of OSCC. Moreover, many published inducible nitric oxide synthase (iNOS), homeobox results showed HER2 expression to be rare in OSCC (5, 12, transcription factor Nanog (NANOG), E-cadherin, and 22). Controversial results in different studies might be due Kangai 1 (KAI-1) in oral cancer. These studies showed that to divergent patient cohorts (location of lesions, gender) or parallel expression of analyzed proteins may exert a differences in IHC methods, type of antibody and tissue synergistic effect on the development, invasion and preparation (5, 13). Similarly to earlier reports (5, 12, 13), metastasis of oral carcinoma (3, 14, 26, 27). Recently, we recorded HER2 gene polymorphism in small number of Kurahara et al. found a correlation between overexpression cases. A high concordance between HER2 by IHC and FISH of p53 protein and PDGFR β expression in pancreatic cancer. observed by other authors (5, 12) was not found in our study. The authors reported that mutant p53 protein overexpression Most likely, our contrary results may be due to methodical induced PDGFR β expression, leading to enhancement of differences and interpretation of IHC staining (5). In oral invasion of pancreatic cancer (28). In the present study, both carcinoma, validation of HER2 IHC and FISH was not PDGFR α and p53 protein expression were correlated. Based performed (5). There are no standardized criteria for IHC and on the study of Kurahara et al. (28), we might suggest that FISH evaluation of HER2 in OSCC (5, 12). Similarly to similar cooperation between PDGFR α and p53 protein exists previous data our results revealed that clinical implication of in oral carcinoma. Relevant to a previous study (29), the HER2 expression/polymorphism in OSCC is limited (5, 12, positive correlation between PDGFR α and HER2 expression 23). Similarly to other reports (5, 24), we did not find a found in this study might suggest that parallel activation of significant relationship between patient age and HER2 these receptors belonging to the same family is possible, overexpression. thereby a paracrine mechanism of activation of co-expressed P53 expression was found in 46.5% of samples, which is receptors might be linked to tumor progression. Interestingly, consistent with previous studies (1, 3, 25). The detection of we found that in poorly differentiated oral carcinomas p53 nuclear p53 accumulation in a large percentage of OSCC in and HER2 expression positively correlated with PDGFR α our study indicates that p53 alteration is a frequent event in expression. Based on earlier published data (3, 14, 26, 27), OSCC (3, 4). Moreover, it is suggested that nuclear and our observations, we may suggest that co-expression of

800 Cierpikowski et al : PDGFR α/ HER2 and PDGFR α/ p53 Co-expression in Oral Carcinoma

PDGFR α/ HER2 or PDGFR α/ p53 might be a biological 9 Schultz JD, Mühlheim K, Erben P Hofheinz RD, Faber A, Thorn feature characterizing a subset of oral carcinomas showing C, Sommer JU, H ӧrmann K and Sauter A: Chemotherapeutic tumor aggressive growth. On the other hand, observed co- alteration of VEGF-/PDGF- and PDGF-R α/β expression by in HPV-transformed squamous cell carcinoma compared expression of PDGFR α/ HER2 and PDGFR α/ p53 in poorly to HPV-negative HNSCC in vitro . Oncol Rep 26 : 1099-1109, 2011. differentiated OSCC indicate these molecules combined with 10 Heldin CH: Targeting the PDGF signaling pathway in tumor morphology of tumor cells might characterize a more treatment. Cell Commun Signal 11 : 97, 2013. invasive phenotype of OSCC. 11 Zhang J, Chen T, Mao Q, Lin J, Jia J, Li S, Xiong W, Lin Y, Liu In conclusion, the findings from this study suggest that Z, Liu X, Zhao H, Wang G, Zheng D, Qiu S and Ge J: PDGFR- cooperation between PDGFR α and HER2 or p53 proteins β- activated ACK1-AKT signaling promotes glioma might lead to more aggressive behavior of OSCC. We propose tumorigenesis. Int J Cancer 136 : 1769-1780, 2015. 12 Birkeland AC, Yanik M, Tillman BN, Scott MV, Foltin SK, that co-expression of PDGRF α/ HER2 or PDGRF α/ p53 in Mann JE, Michmerhuizen NL, Ludwig ML, Sandelski MM, poorly differentiated OSCC indicates that these proteins may Komarck CM, Carey TE, Prince ME, Bradford CR, McHugh JB, cooperate in invasion and enhance progression of OSCC. Spector ME and Brenner JC: Identification of targetable ERBB2 aberrations in head and neck squamous cell carcinoma. JAMA Conflicts of Interests Otolaryngol Neck Surg 142 : 559-567, 2016. 13 Dalal V, Alam K, Maheshwari V, Sharma S, Jain A and Khan R: None to declare. Correlation of clinicopathological parameters with EGFR and HER-2 neu status in oral cancer patients in a tertiary care centre. Acknowledgements Int J Biomed Res 6: 897-902, 2015. 14 Yang L, Wang Y, Guo L, Wang L, Chen W and Shi B: The expression and correlation of iNOS and p53 in oral squamous The Authors offer special thanks to Dr Piotr Grelewski for his cell carcinoma. Biomed Res Int 2015 : 1-8, 2015. excellent technical assistance. This study was supported by Wroclaw Medical University (grant no. ST.B132.16.052). 15 Dave K, Chalishazar M, Dave V, Panja P, Singh M and Modi T: Immunohistochemical expression of p53 and its clinicopathological correlation with modified Anneroth’s histological grading system. References J Oral Maxillofac Pathol 20 : 29-35, 2016. 16 Barnes L, Eveson JW, Reichart P and Sidransky D: World Health 1 Cutilli T, Leocata P, Dolo V and Altobelli E: p53 as a prognostic Organization Classification of Tumours: Pathology and Genetics marker associated with the risk of mortality for oral squamous of Head and Neck Tumours. Lyon, IARC Press, 2005. cell carcinoma. Oncol Lett 12 : 1046-1050, 2016. 17 Edge SB and Compton CC: The American Joint Committee on 2 Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J and Cancer: the 7th Edition of the AJCC Cancer Staging Manual and Jemal A: Global cancer statistics, 2012. CA Cancer J Clin 65 : the future of TNM. Ann Surg Oncol 17 : 1471-1474, 2010. 87-108, 2015. 18 Wolff AC, Hammond ME, Hicks DG, Dowsett M, McShane 3 Lee HJ, Kang YH, Lee JS, Byun JH, Kim UK, Jang SJ, Rho GJ LM, Allison KH, Allred DC, Bartlett JM, Bilous M, Fitzgibbons and Park BW: Positive expression of NANOG, mutant p53, and P, Hanna W, Jenkins RB, Mangu PB, Paik S, Perez Ea, Press CD44 is directly associated with clinicopathological features and MF, Spears PA, Vance GH, Viale G and Hayes DF: poor prognosis of oral squamous cell carcinoma. BMC Oral Recommendations for Human Epidermal Growth Factor Health 15 : 153, 2015. Receptor 2 Testing in Breast Cancer: American Society of 4 Zedan W, Mourad MI, El-Aziz SMA, Salamaa NM and Shalaby Clinical Oncology/College of American Pathologists Clinical AA: Cytogenetic significance of chromosome 17 aberrations and Practice Guideline Update. J Clin Oncol 31 : 3997-4013, 2013. P53 gene as prognostic markers in oral squamous cell 19 Steller EJ, Raats DA, Koster J, Rutten B, Govaert KM, Emmink carcinoma. Diagn Pathol 10 : 2, 2015. BL, Snoeren N, van Hooff SR, Holstege FC, Maas C, Borel 5 Hanken H, Gaudin R, Gröbe A, Fraederich M, Eichhorn W, Rinkes IH and Kranenburg O: PDGFRB promotes liver Smeets R, Simon R, Sauter G, Grupp K, Izbicki JR, Sehner S, metastasis formation of mesenchymal-like colorectal tumor cells. Heiland M and Blessmann M: HER2 expression and gene Neoplasia 15 : 204-217, 2013. amplification is rarely detectable in patients with oral squamous 20 Sardari Y, Pardis S, Tadbir AA, Ashraf MJ, Fattahi MJ, Ebrahimi H, cell carcinomas. J Oral Pathol Med 43 : 304-308, 2014. Pursahidi S, Khademi B and Hamzavi M: HER2/neu expression in 6 Iqbal N and Iqbal N: Human epidermal growth factor receptor head and neck squamous cell carcinoma patients is not significantly 2 (HER2) in cancers: overexpression and therapeutic elevated. Asian Pac J Cancer Prev 13 : 2891-2896, 2012. implications. Mol Biol Int 2014 : 1-9, 2014. 21 Khademi B, Shirazi FM, Vasei M, Doroudchi M, Gandomi B, 7 Carrington C: Oral targeted therapy for cancer. Aust Prescr 5: Modjtahedi H, Pezeshki AM and Ghaderi A: The expression of 171-176, 2015. p53, c-ERBB-1 and c-ERBB-2 molecules and their correlation 8 Aderhold C, Umbreit C, Faber A, Sauter A, Sommer JU, Birk R, with prognostic markers in patients with head and neck tumors. Erben P, Hofheinz RD, Stern-Straeter J, H ӧrmann K and Schultz Cancer Lett 184 : 223-230, 2002. JD: Chemotherapeutic alteration of VEGF, PDGF and 22 Dragomir LP, Mărgăritescu C, Florescu A, Olimid AD, Dragomir PDGFR α/β expression under 5-FU vs. docetaxel in HPV- M and Popescu MR: The immunoexpression of EGFR and transformed squamous cell carcinoma compared to HPV- HER2/neu in oral squamous carcinoma. Rom J Morphol negative HNSCC in vitro . Anticancer Res 33 : 1951-1961, 2013. Embryol 53 : 597-601, 2012.

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23 Pollock NI and Grandis JR: HER2 as a therapeutic target in head 28 Kurahara H, Maemura K, Mataki Y, Sakoda M, Shinchi H and and neck squamous cell carcinoma. Clin Cancer Res 21 : 526- Natsugoe S: Impact of p53 and PDGFR- β expression on 533, 2015. metastasis and prognosis of patients with pancreatic cancer. 24 Tse GM, Yu KH, Chan AW, King AD, CHen GG, Wong KT, World J Surg 40 : 1977-1984, 2016. Tsang RK and Chan AB: HER2 expression predicts improved 29 Kartha VK, Stawski L, Rong H, Paul H, Gallagher G, Noonan survival in patients with cervical node-positive head and neck V, Kukuruzinska M, Monti S and Trojanowska M: PDGFR β is squamous cell carcinoma. Otolaryngol Neck Surg 141 : 467-473, a novel marker of stromal activation in oral squamous cell 2009. carcinomas. PLoS One 11 : e0154645, 2016. 25 Li L, Fukumoto M and Liu D: Prognostic significance of p53 immunoexpression in the survival of oral squamous cell carcinoma patients treated with surgery and neoadjuvant . Oncol Lett 6: 1611-15, 2013. 26 Fan CC, Wang TY, Cheng YA, Jiang SS, Cheng CW, Lee AY and Kao TY: Expression of E-cadherin, TWIST, and p53 and their prognostic value in patients with oral squamous cell carcinoma. J Cancer Res Clin Oncol 139 : 1735-1744, 2013. 27 Patil NN, Wadhwan V, Chaudhary M and Nayyar AS: KAI-1 and p53 expression in oral squamous cell carcinomas: Markers of Received November 3, 2017 significance in future diagnostics and possibly therapeutics. J Revised November 25, 2017 Oral Maxillofac Pathol 20 : 384-389, 2016. Accepted November 28, 2017

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