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Pickhard et al., J Otol Rhinol 2012, 1:2http://dx.doi.org/10.4172/2324-8793.1000104

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Expression of STK15, EGFR, p53 and p16INK4A by Squamous Cell Carcinoma (SCC) of the Nasal Cavity and the Paranasal SinusesAnja C. Pickhard1*, Guido Piontek1, Andreas Knopf1, Thomas Stark1, Anne-Laure Boulesteix2, Rainer Staudenmaier1 and Rudolf Reiter3

AbstractObjective: Squamous cell carcinoma (SCC) of the nasal

cavity and the paranasal sinuses is very rare and therefore poor understood. The purpose of our study was to investigate expression patterns of STK15, EGFR, p53 and p16 in these tumor samples and to correlate with histomorphological findings and clinical follow-up data.

Patients and methods: The expression of STK15, EGFR, p53 and p16 was studied in a series of 22 tumor samples of patients with primarily resected SCC of the nose. The expression analysis on protein level was performed on paraffin fixed and wax-embedded material by immunhistochemical staining. The results were correlated with clinicopathological features and survival data.

Results: Expression of STK15 was observed in 14 of 21 cases (67%), EGFR expression in 16 of 18 cases (89%) and accumulation of p53 in 16 of 21 cases (76%). Loss of p16 expression was observed in 8 of 21 cases (38%). There was no positive correlation with clinicopathological features and survival data.

Conclusion: Identification of any marker superior to or even approaching the prognostic value off conventional histopathological markers was therefore not possible.

Keywords: Nasal squamous cell carcinoma; STK15; EGFR; p53; p16

IntroductionCancers of the nasal cavity or the paranasal sinuses have a low

prevalence and incidence. In addition the presentation in various subsites of the nose and the paranasal sinuses and the existence of multiple tumor histopathologies complicate an execution of a study. These cancers represent 0.5% of all malignant neoplasms and 3% of head and neck cancers [1]. 50% of the nasal cancers are squamous cell carcinomas (SCC). Tumors such as adenocarcinoma, melanoma and adenoid cystic carcinoma constitute the remaining 50% [2-4]. Treatment options for nasal cavity or paranasal sinuses cancer

*Corresponding author: Anja C. Pickhard, Klinikum rechts der Isar, Hals-Nasen-Ohren-Klinik, Ismaninger Str. 22, 81675 München, Germany, Tel: -49-(0)89-41405319; Fax: -49-(0)89-41404952; E-mail: a.pickhard@lrz.tum.de

Received: August 09, 2012 Accepted: September 24, 2012 Published: September 26, 2012

include surgery, radiation therapy, or a combination of the two [5]. A recent large analysis reported 5-year survival and local control rates of 40% and 59%, respectively [1].

In the last decade significant progress has been made in the understanding of the molecular mechanisms, which are responsible for human cancer development and progression.

STK15 also known as Aurora-A/AURKA/BTAK/AIK1 is a member of the Aurora/Ipl1p family of cell cycle–regulating Serine/Threonine kinases. The up-regulation of STK15 leads to abnormal centrosome numbers and the induction of aneuploidy [6,7], which is a very frequent event in head and neck squamous cell carcinoma (HNSCC), found in up to 90% [8]. A correlation between the up-regulation of STK15 and clinical aggressiveness has been described for several cancers [9-13]. Previously, we demonstrated a correlation between the overexpression of STK15 and poor clinical outcome of patients with HNSCC [9].

The epidermal growth factor receptor (EGFR), a member of the structurally related erbB family of receptor tyrosine kinase, has been implicated in cancer development and progression in a large number of tumors including HNSCC [14].

Today it is clear that the p53 pathway plays an important role in HNSCC biology and potentially in its treatment. Patients with HNSCC having a high portion of tumor cells expressing p53 had a shorter survival than the other groups [15]. Overexpression of p53 protein was also identified in nasal cancer. However, approximately 60% of the nasal cancer shows this overexpression [16,17].

Functional inactivation of p16 is known to be a common event in HNSCC, mainly by either deletion or methylation [18]. p16 is an inhibitor of cyclin-dependent kinases 4 and 6, which activate the negative cell cycle regulator protein pRB which in turn down regulates p16 expression. It could be shown that in cervix uteri tumors E7 protein of the high-risk HPVs can interfere with this regulatory circuit by its virtue to inactivate pRB and thus lead to the overexpession of p16. Furthermore, p16 expression in HNSCC correlates with HPV positive tumors [19]. While HPV-free and non-transcriptionally active HPV-related patients showed similar 5-years survival rates, E6/E7 expression was associated with a better prognosis [20].

Squamous cell carcinoma of the nasal cavity and the paranasal sinuses are very rare and thus immunostaining studies barely exist. In addition, in many reviews of nasal cancer, all different histopathologies are analyzed together. The purpose of our study was first to investigate expression patterns of STK15, EGFR, p53 and p16 in SCC of the nasal cavity and paranasal sinuses and second to investigate a correlation with histomorphological findings and clinical follow-up data of the patients.

Material and MethodsPatient selection and tissue samples

Paraffin wax-embedded tumor samples from 22 patients (mean age 60 years, range 41-79 years) with a squamous cell carcinoma of the nasal cavity (n=17) or of the paranasal sinuses (n=5) were investigated.

Citation: Pickhard AC, Piontek G, Knopf A, Stark T, Boulesteix AL, et al. (2012) Expression of STK15, EGFR, p53 and p16INK4A by Squamous Cell Carcinoma (SCC) of the Nasal Cavity and the Paranasal Sinuses. J Otol Rhinol 1:2.

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Patients had been treated by radical surgical resection between 2004 and 2009 in the Department of Head and Neck Surgery, Klinikum rechts der Isar, Technical University Munich. If there were suspected lymph nodes in the x-ray or ultrasonography, a neck dissection was performed. In case of lymph node metastasis or incomplete resection of the tumor patients received adjuvant radiotherapy. The pT and pN categories of the tumor were determined according to the current tumor-node-metastasis classification [21] and tumor grading according to the World Health Organization (WHO) classification [22].

For all tumors, histopathological and clinical follow-up data were available from follow-up examinations (follow-up period of 6 to 60 months). Clinical data (site of primary tumor, tumor and nodal clas-sification, histological grade) from the patients were retrieved from medical records (Table 1) and survival data (disease free survival (DFS) and overall survival (OS)) were evaluated by the Tumorregister München (Institut für medizinische Informationsverarbeitung, Bi-ometrie und Epidemiologie (IBE), Ludwig-Maximilians-University, Munich). These findings were correlated with expression patterns of STK15, EGFR, p53 and p16.

The study was approved by the Medical Ethics Committee of the Technical University of Munich. Detailed patient characteristics and histomorphological features are shown in table 1.

Immunhistochemical study

Immunhistochemistry was performed on deparaffinized tissue sections (2µm), stained with antibodies against STK15 (Novocastra, Leica-Microsystems, Wetzlar, Germany/dilution 1:100), EGFR (Santa Cruz Biotech, Santa Cruz, USA/dilution 1:200), p53 (DAKO, Hamburg, Germany/dilution 1:200) and p16INK4A (NeoMarkers, Fremont, USA/dilution 1:100), visualized with peroxidase-conjugated secondary antibody (LSAB Kit, DAKO, Hamburg, Germany) (Table 2). The tissue sections were counterstained with Mayer hematoxylin solution. For positive controls, we used tissues with known expression of the respective antigens. For negative controls, we used irrelevant antibodies with the immunoglobulin isotype.

Scoring

According to previously published criteria cytoplasmatic and/or nuclear immunoreactivity of STK15 [9], p53 and p16 [23] and the membrane and/or cytoplasmatic staining of EGFR [24] was evaluated in three tumor areas of each case (Figure 1).

Immunoreactivity of STK15 was scored into five groups according to the percentage and intensity of cytoplasmatic and/or nuclear staining of the positively stained tumor cells [9]. Specimens with >30% of cells stained were scored as strongly positive (3+), those with 10-30% of cells stained were scored as moderately positive (2+), those with <10% cells stained were scored as weakly positive (1+). Specimens with no staining were scored as negative.

EGFR membrane staining was quantified and graded as described recently [24]: no staining or membrane staining in <10% neoplastic cells: negative (score: 0); complete or incomplete membranous staining in >10% neoplastic cells: positive (weak staining: score 1+; moderate staining: score 2+, strong staining: score 3+).

In cases of p53 and p16 staining, a visual grading system based on the number of positively stained nuclei of the malignant cells in each tissue was used: >10% nuclei stained: positive, irrespective of staining intensity [23].

All scoring analysis was done by two independent investigators.

Statistical analysis

The association between survival and expression patterns for each marker, pT classification, grade and localization was statistically assessed based on the logrank test and represented graphically using Kaplan-Meier curves. Relationships between the four proteins or between the proteins and pT classification, grade or localization were tested by crosstabs and the exact Fisher test.

All statistical analyses were performed using the R software. Results with p-values <0.05 were considered significant.

Figure 1: Patients with lower grade tumor and a pT1-2 classification show a longer survival..

T1-2T4

Kaplan-MelerpT classification T1-2 vs. T4

Sur

viva

l pro

babi

lity

Survival time (in days)0 500 1000 1500

p-value=0.054

0.0

0.2

0.

4

0.6

0.8

1.

0

Characteristics Data, n (%)

SexFemale 8 (36)

Male 14 (64)

LocalizationNasal cavity 17 (77)

Paranasal sinuses 5 (23)

TherapySurgery alone 16 (73)

Surgery+radiation 6 (27)

pT category

pT1 11 (50)pT2 7 (32)pT3 0 (0)

pT4a 4 (18)

c/pN categoryc/pN0 22 (100)pN+ 0 (0)

Grade

G1 2 (9)G2 11 (50)G3 8 (36)G4 1 (5)

Table 1: Clinicopathological features of 22 patients with squamous cell carcinomas of the nasal cavity or the paranasal sinuses.

Citation: Pickhard AC, Piontek G, Knopf A, Stark T, Boulesteix AL, et al. (2012) Expression of STK15, EGFR, p53 and p16INK4A by Squamous Cell Carcinoma (SCC) of the Nasal Cavity and the Paranasal Sinuses. J Otol Rhinol 1:2.

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ResultsImmunostaining patterns

Neoplastic tissue was interpretable for all 22 cases. Expression of EGFR was observed in 16 of 18 cases (89%), accumulation of p53 in 16 of 21 cases (76%) and of STK15 in 14 of 21 cases (67%). Loss of p16 expression was observed in 8 of 21 cases (38%) (Table 3).

Association with survival

We found no significant association between the expression patterns for STK15, EGFR, p53, p16 and survival (STK15>0: p=0.72, EGFR>1: p=0.24, p53>0: p=0.23, p16>0: p=0.55).

Also pT classification (T1-2-4: p=0.15; T1-2 vs. T4: p=0.05), grade (G>1: p=0.13) and localization (p=0.25) were not significantly associated with survival in our population. But patients with lower grade tumor and a pT1-2 classification show a longer survival (Figures 1 and 2).

Furthermore, there was no significant correlation between therapy and survival (p=0.95).

Relationships between proteins and clinicopathological features

For the protein expression level, there were a relationships among EGFR and p16 (p=0.019). The other proteins show no relationship. Also, we found no association between the four proteins and pT classification, grade or tumor localization (p>0.05).

DiscussionIn this report, we described the first systematic survey of STK15,

EGFR, p53 and p16 expression in SCC of the nasal cavity and the paranasal sinuses. The results showed that protein up-regulation,

especially STK15, EGFR and p53, frequently occurs in SCC. We did not found a correlation with survival.

STK15 is a member of the Aurora/Ipl1p family of cell cycle–regulating Serine/Threonine kinases and is localized at interphase and mitotic centrosomes and the spindle poles where it regulates proper chromosome segregation and cytokinesis [25]. Furthermore, the up-regulation of STK15 leads to abnormal centrosome numbers and the induction of aneuploidy [6,7], which is a very frequent event in HNSCC, found in up to 90% [8]. A correlation between

the up-regulation of STK15 and clinical aggressiveness has been described for several cancers [9-13]. These findings suggest that STK15 is a critical kinase-encoding gene whose up-regulation leads to centrosome amplification and chromosomal instability, indicating its involvement in tumorigenesis also in SCC of nasal cavity and the paranasal sinuses.

Recent studies have described a correlation between the up-regulation of STK15 with tumor size, progression and clinical aggressiveness in several cancer types [10-13], which could be also shown in our series of HNSCC patients [9]. In this study of SCC of nasal cavity and the paranasal sinuses we found no correlation of STK15 expression and survival data.

EGFR activation results in a cascade of cellular responses occurring in cell division, proliferation, differentiation, apoptosis and angiogenesis. The prognostic significance of EGFR expression has been established for various tumor entities including HNSCC [14]. EGFR over-expression occurs early in the pathogenesis of HNSCC [26] and is associated with reduced relapse-free survival or poor overall survival time [27]. At clinical level, inhibition of EGFR with monoclonal antibody showed potential therapeutic effects with better survival of patients when added to standard radiotherapy [28]. Also, it was shown, that positive rates of immunostaining for EGFR protein were significantly up-regulated in carcinoma of the nasal cavity when comparing polyp and normal mucosa [29]. In contrast, we could not show a correlation with EGFR expression and clinicopathological criteria. Nevertheless, we saw an over-expression of the EGFR in

Marker Clone Company Dilution Buffer, antigen retrieval

EGFR 1005 Santa Cruz Biotech 1:200 Citrat pH 6.0

STK15 JLM Novocastra 1:100 Citrat pH 6.0

p16 16P07 NeoMarkers 1:100 Citrat pH 6.0

p53 D07 Dako Cytomation 1:200 Citrat pH 6.0

Table 2: Antibodies used in this study.

Marker Staining patterns Fraction of staining patterns (%)

STK15

negative 7/21 (33)positive 14/21 (67)score 1+ 10/21 (47)score 2+ 2/21 (10)score 3+ 2/21 (10)

p53negative 5/21 (24)positive 16/21 (76)

EGFR

negative 2/18 (11)positive 16/18 (89)score 1+ 4/18 (22)score 2+ 3/18 (17)score 3+ 9/18 (50)

p16negative 13/21 (62)positive 8/21 (38)

Table 3: Immunhistochemical expression patterns of STK15, p53, EGFR and p16 in 22 patients with squamous cell carcinomas of the nasal cavity or the paranasal sinuses.

Figure 2: Patients with lower grade tumor and a pT1-2 classification show a longer survival..

Survival time (in days)S

urvi

val p

roba

bilit

y

0 500 1000 1500

0.0

0.2

0.4

0.

6

0

.8

1

.0

G1-2G3-4

p-value=0.132

Kaplan-Melergrade G1-2 vs. G3-4

Citation: Pickhard AC, Piontek G, Knopf A, Stark T, Boulesteix AL, et al. (2012) Expression of STK15, EGFR, p53 and p16INK4A by Squamous Cell Carcinoma (SCC) of the Nasal Cavity and the Paranasal Sinuses. J Otol Rhinol 1:2.

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89% of the cases. Maybe a monoclonal antibody therapy (against the EGFR) could also be an option for patients with a SCC of the nose as observed for patients with HNSCC [30].

In the present study, 76% of the nasal cancer showed positive immunostaining for p53. This was also observed by Fang et al. [16,17]. Many recent studies have focused on the TP53 tumor suppressor gene, analyzing its gene and protein status. When looking at p53 protein expression, using immunohistochemistry, no correlation with patient outcome has been seen for the whole group of HNSCC. Concerning the prognostic significance of mutations in the TP53 gene, results differ. But when restricting analysis to tumors with mutations causing an obvious change in protein, TP53 mutation was found to be a strong and independent variable for prognosticating survival [31]. Today it is obvious that the p53 pathway is very important in HNSCC biology and potentially in its treatment. Patients with HNSCC having a high portion of tumor cells expressing p53 had a shorter survival than the other groups [15]. Expression of p53 was found in 71.7% of HNSCC and a higher expression was noted in lesions of young patients [32].

Loss or inactivation of p16 occurred in 8 cases (38%) in our study. In contrast, El-Mofty et al. found a negative or weakly reactive to p16 antibodies in SCC of the nasal cavity [33]. Functional inactivation of p16 is known to be a common event in HNSCC, mainly by either deletion or methylation [18]. The incidence of HPV induced oropharyngeal squamous cell carcinoma (OPSCC) increases in the western countries. These OPSCC show distinct molecular characteristics and are characterized by an overexpression of p16, considered a surrogate marker for HPV infection. Compared to patients with p16 negative OPSCC, patients with HPV induced p16 positive OPSCC show a significantly better prognosis, which is reported to be due to increased radiosensitivity [34].

A limitation of our investigations was the small number of patients, but squamous cell carcinoma (SCC) of the nasal cavity and the paranasal sinuses is very rare and therefore poorly understood. Our study should give a first indication for further analysis.

In conclusion, our preliminary findings showed that STK15, EGFR and p53 protein up-regulation is a common abnormality in SCC of nasal cavity and the paranasal sinuses. It was disappointing, however, that none of the molecular markers analyzed in this small study were of prognostic value. But for the study of the possible prognostic value of tumor markers a larger multicenter study is needed.References

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Citation: Pickhard AC, Piontek G, Knopf A, Stark T, Boulesteix AL, et al. (2012) Expression of STK15, EGFR, p53 and p16INK4A by Squamous Cell Carcinoma (SCC) of the Nasal Cavity and the Paranasal Sinuses. J Otol Rhinol 1:2.

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Author Affiliations Top1Department of Otolaryngology Head and Neck Surgery, Technical University of Munich, Ismaninger Straße 22, D-81675 Munich, Germany2Department of Medical Informatics, Biometry and Epidemiology, Ludwig-Maximilians-University Munich, Marchioninistr. 15, 81377 Munich, Germany3Department of Otolaryngology Head and Neck Surgery, Section of Phoniatrics and Pedaudiology, University of Ulm, Prittwitzstr. 43, 89070 Ulm, Germany