Impact of HPV Status and AurkA Phe31Ile Polymorphism on The Response to Cetuximab Treatment of Head and Neck Squamous Cell Carcinoma (HNSCC) Patients

Background: Since 2004, the use of the monoclonal antibody cetuximab has been preferred over platinum-based therapy for patients with advanced Head and neck squamous cell carcinoma (HNSCC). However, the response rate to the treatment is only around 20%. Currently, no biomarkers have been identied to differentiate potential responders from non-responders to cetuximab therapy. Methods: We evaluated the predictive and prognostic properties of AurkA polymorphism and HPV infection in HNSCC patients treated with cetuximab. Clinical data of 434 patients was collected and tissue was analyzed for AurkA polymorphism using PCR. Immunohistochemistry was used to stain for various markers and their expression levels were scored. Cell culture experiments were performed to complement clinical ndings. Results: We demonstrated in vivo as well as in vitro that both AurkA polymorphism and HPV status have predictive and prognostic value. Conclusions: AurkA polymorphism and HPV status could be benecial for response prediction and therapy optimization for HNSCC patients. DNA was isolated from cell lines and FFPE tumor samples using the DNeasy Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Specic PCR for AurkA genotypes at the Phe31Ile site was performed using the Taq-DNA Polymerase All-inclusive Kit (PeqLab, Erlangen, Germany) according the manufacturer’s guidelines. We applied the following PCR program: 95 °C for 5 min, 30 cycles of 95 °C for 30 s, 60 °C annealing for 30 s, 72 °C for 30 s, and 72 °C for 7 min on a thermocycler (BioRad, Munich, Germany). The following primers were used: AurkA forward: CTTTCATGAATGCCAGAAAGTT; AurkA reverse: CTGGGAAGAATTTGAAGGACA. The 165-bp PCR products were digested with ApoI (New England BioLabs, Inc., Beverly, USA) and separated on a 2.5% agarose gel. Digestion of the AurkA 31Phe allele results in two fragments (153 bp and 12 bp); since the AurkA 31Ile allele contains an additional third ApoI restriction site, three fragments (89 bp, 64 bp, and 12 bp) are generated after digestion. The results were further conrmed by DNA sequencing using an ABI 3100 DNA sequencer (Life Technologies, Darmstadt, Germany).

Cetuximab is a chimeric (65% human and 35% mouse) monoclonal IgG1 antibody directed against the extracellular binding domain of the EGFR [21]. The antibody competes with the receptor for ligand binding and upon binding causes receptor internalization and degradation [22,23].
Aurora kinases (Aurks) were described in 1995 by David Glover et al. [24,25]. Aurks are cell cycle regulating serine-threonine kinases. In humans, the Aurk family comprises three members: Aurora kinase A, B, and C [26]. Aurora kinases A and B play pivotal roles in the regulation of cell division, especially during entry into mitosis by the formation of a functional microtubule spindle apparatus and in the completion of cytokinesis [27]. Overexpression of Aurora kinase A exists in tissues with a high mitotic index, e.g., in the thymus or embryo, as well as in many tumor types, including carcinomas of the colon, breast, stomach, ovary, non-small cell lung carcinoma, and in squamous cell carcinoma of the head and neck [28-30, 27, 31-33]. Overexpression in tumor tissues is usually caused by AurkA gene ampli cation [34]. Recently, the ampli cation of chromosome 20q has been observed in a wide variety of tumor types as an important mechanism of enhanced AurkA gene dosage [35,36]. Overall, an increased number of AurkA genes seem to be clinically associated with more aggressive cancer, independent of the molecular mechanism that causes an enhanced gene dose [37,33].
An AurkA genetic polymorphism has been described, in which the amino acid phenylalanine (Phe) at position 31 is replaced by isoleucine (Ile) as a result of different coding regions [38]. In the case of esophageal carcinoma, the Phe31Ile polymorphism has been classi ed as a risk factor for both tumor occurrence and progression [31]. Since 2004, the therapeutic application of the monoclonal antibody cetuximab has been preferred over platinum-based chemotherapy for the treatment of patients with advanced HNSCC. We have previously shown that in vitro, the response rate to cetuximab treatment was higher in patients with the homozygous polymorphism [39].

Methods
Aim, design and setting of the study Response to cetuximab therapy in patients with HNSCC is only around 20%, and currently, neither prognostic nor predictive biomarkers have been identi ed to stratify patients with respect to potential response vs. non-response to cetuximab.
The aim of the present study was to investigate the HPV status, and in particular the Aurora kinase A polymorphism, in terms of their predictive and prognostic value in patients with HNSCC treated with cetuximab therapy.
Clinical data from patients with HNSCC was retrospectively analyzed. In vitro experiments were performed to complement clinical data collected.
Clinical data and experimental procedures Patient population FFPE tissues from 434 patients with squamous cell carcinoma of the oral cavity, oropharynx, hypopharynx, and larynx were used. Both tumor tissue and corresponding normal tissue from the same patient were examined. The included patients were treated at the ENT hospital, Klinikum rechts der Isar, Technical University Munich, at the ENT department of the University of Regensburg and the ENT department of the University of Heidelberg. The patients received either surgery, primary chemoradiotherapy (combined with cetuximab in many cases), or palliative chemo-or immunotherapy.
The inclusion criterion was admittance to one of the three hospitals from 1988 to 2015. Histological and clinical data were collected from medical records, and information from the Munich Cancer Registry was used for the collection of survival data. Patient characteristics are enumerated in Table 1  Immunohistochemical study Fresh 1.5 µm sections were transferred to glass slides, depara ned, and rehydrated. An antigen retrieval method (microwave activation in citrate buffered saline) was applied following the instructions provided by the manufacturer. After cooling, the slides were incubated with the following antibodies: Aurora-Kinase Scoring system for protein expression in immunohistochemical staining A scoring system was applied to describe the expression levels of the proteins AurkA, AurkB, Survivin, and p-Akt Ser473. The stained tumor areas were dichotomized as follows: Adopted from Schauer et al., we used an immunostaining score comprised of intensity and a stained tumor area that had values between 0 and 7 [40]. To perform the statistical analysis, we set a cut-off at 2 and divided the samples into positive and negative. p16 INK4a was considered to be positive when it was de ned as strong and diffuse nuclear and cytoplasmic staining in ≥ 70% of the tumor cells, which is the same scoring criteria used in the study by Ang et al. [41].
Cell culture

Analysis of cell proliferation
The binding of crystal violet to cellular DNA was used to assess cell proliferation via photometry. Cells were seeded at a density of 5 × 10 3 cells per well in six-well plates 24 h before treatment. Ten days after treatment with the inhibitors, the culture medium was aspirated and 500 µL of 4% formaldehyde was added to each well for 15 minutes. After washing with 0.1% Triton-X100/1x PBS and H 2 O, crystal violet (0.04%) was added to the xed cells and incubated for 30 minutes. Finally, after removing excess crystal violet, SDS (1%) was added, and the optical density was measured at 595 nm using a microplate photometer after 1 h.

Colony formation assay
Cell survival after treatment with inhibitors was assessed using a colony formation assay (CFA). The cells were seeded in six-well plates (5 × 10 2 cells per well) and allowed to adhere overnight at 37 °C. The following day, the cells were treated and incubated at 37 °C for 10 days. The cell colonies were then formalin xed (4% formaldehyde) and visualized by crystal violet (0.04%) staining (Sigma-Aldrich, Steinheim, Germany). The colonies were counted after rinsing off the dye. Colony numbers were depicted as the percentage of colonies from untreated cells.

Aneuploidy analysis by ow cytometry
After seeding in culture asks (75 cm

Proliferation assay
Both HPV positive cell lines were very sensitive to the individual treatments. The strongest antiproliferative effect following treatment with cetuximab monotherapy, as well as the combination treatment, was observed in the HPV positive and AurkA heterozygous cell line UP-SCC-154. The combined application of cetuximab and docetaxel was even more e cient at inhibiting cell proliferation than the application of either substrate alone (p = 0.0017). The analysis of the Aurora kinase A heterozygous cell line SAS tended to show the same phenomenon.
Neither treatment with cetuximab alone nor the combination of cetuximab and docetaxel caused an e cient inhibition of the HPV negative AurkA homozygous cell line UD-SCC-5. Only cetuximab treatment resulted in a signi cant reduction in the survival of the SAS cell line.
Using advanced analysis we grouped and evaluated the results depending on the property. The results demonstrated that treatment with cetuximab caused a signi cantly more e cient response in HPV positive than in HPV negative cells (Fig. 1).
The antiproliferative effect was even more pronounced in AurkA heterozygotes compared to AurkA homozygous cell lines.
The most e cient inhibition of proliferation, by either single or combination treatments, was found in cells harboring both a heterozygous AurkA polymorphism and HPV infection. Combined treatment with cetuximab and docetaxel was even more e cient in HPV positive AurkA heterozygous cell lines than the application of cetuximab alone (p = 0.0017). This phenomenon appeared only as a trend in the AurkA heterozygous cell lines with HPV negative status (p = 0.0929). In contrast, in cells with homozygous AurkA polymorphism, regardless of the HPV status, no increased inhibition of proliferation by the additional use of docetaxel was observed.

Colony formation assay
In both HPV positive cell lines UD-SCC-2 and UP-SCC-154 the formation of proliferative colonies could be signi cantly reduced by the use of cetuximab alone or in combination with docetaxel, regardless of the Aurora kinase A polymorphism.
In HPV negative cell lines, the antiproliferative effect was signi cantly lower, while the UD-SCC-5 did not respond to the treatments at all.
In order to better evaluate the in uence of the examined characteristics on the clonogenic survival of the cell lines, the results were regrouped and reevaluated depending on the property. The analysis revealed that the HPV positive cells were mostly sensitive to the treatments (Fig. 2).
In contrast, the application of cetuximab alone or in combination with docetaxel did not elicit any effect on the clonogenic survival of HPV negative cells.
Both cetuximab monotreatment and the combination treatment signi cantly reduced clonogenic formation of the AurkA homozygous and AurkA heterozygous cell lines, respectively (Fig. 2). However, there was no signi cant difference in terms of effect as a function of treatment in the group comparison.
In terms of the inhibition of clonogenic survival, overall, the most pronounced treatment e ciency with respect to the clonogenic survival was observed when cells with heterozygous Aurora kinase A polymorphism were positive for HPV. In the heterozygous cell lines the combined use of cetuximab and docetaxel was more e cient compared to the application of cetuximab alone (AurkA heterozygous and HPV positive, p = 0.0852; AurkA heterozygous and HPV negative, p = 0.0765).

Western blot analysis
We evaluated Aurora kinase A and B, p-Akt Ser473, as well as Survivin expression on the protein level as a function of single (either cetuximab or docetaxel) and dual (combined cetuximab and docetaxel) treatment (Fig. 3) The analysis revealed higher protein levels of Survivin, p-Akt Ser473, and Aurora kinase B in the heterozygous cell lines compared to the homozygous cell lines. Treatment of the cells, in particular with the combination of cetuximab and docetaxel, led to a reduction of AurkA protein expression in the HPV negative cell lines. The strongest effect was observed in the homozygous UD-SCC-5 cell line.

Ploidy analysis
In order to show the relationship between the Aurora kinase A polymorphism and the potential genesis of polysomy/aneuploidy, the total cellular DNA content of the cells was determined by ow cytometry.

Overall survival
By Kaplan-Maier analyses we veri ed prolonged overall survival for the entire collective, and especially for HPV positive patients under cetuximab therapy (p = 0.001) (Fig. 6).
Furthermore, the clinical data revealed a signi cantly longer survival for patients with homozygous Aurora kinase A (p = 0.048). The HPV status, as well as the Aurora kinase A polymorphisms, seem to play an important role in terms of therapeutic response to cetuximab treatment. The in vivo analysis revealed a positive trend for patients harboring a homozygous Phe/Phe Aurora kinase A.

Discussion
Due to the stable overexpression of EGFR in HNSCC, the introduction of anti-EGFR therapy with cetuximab in patients with HNSCC seemed very promising [42]. Yet, among other aws, the response rate in patients only reaches around 20% [19]. Due to the complexity of the signaling pathways and the large number of proteins involved, the quest for predictive biomarkers for the response to cetuximab therapy remains inconclusive [13,43].
Here we assessed the HPV infection status and the Aurora kinase A polymorphism as potential markers for cetuximab treatment e cacy.
The clinico-pathological characteristics of the HNSCC patient collective analyzed in this study were comparable to those reported elsewhere. Approximately 80% of all patients were male and the primary tumors were most often located in the oropharynx. Nicotine and alcohol abuse are known as the two most important risk factors for the development of HNSCC, which is also re ected by the demographic data of the study collective [44,45].
Unlike in the US for instance, in Germany no standard therapy concept was carried out by default in the described collective over the observed time period. For each patient the treatment was rather adjusted individually, according to the stage of the disease, general condition, and patient's will. Hence, the treatment strategies to compare were heterogeneous and thus the number of cases in the individual groups were often very low.

Comparison of the expression of Aurora kinases, p-Akt Ser473 and Survivin
The rst evidence that Aurora kinase A is overexpressed in solid tumors was reported in 1998 [29,46].
Accordingly, increased aurora kinase A protein levels could also be detected in the tumor tissue of HNSCC patients [27]. Here, we observed a signi cantly higher expression of Aurora kinase A in the tumor tissue compared to the corresponding normal tissue (p < 0.0001).
Consistent with the literature, a signi cantly elevated level of p-Akt Ser473 (p = 0.014) and Survivin (p = 0.003) in the tumor tissue was also recorded in the present patient collective [47][48][49][50][51]. It is well known that high levels of these proteins cause chromosomal instability and therefore contribute to carcinogenesis as an early hallmark [52][53][54].
Survivin not only plays a central role as an inhibitor of apoptosis but is also an integral part of the chromosomal passenger complex (CPC) [50]. Together with other proteins of the CPC, such as Aurora kinase B, Survivin is also involved in the regulation of cell division [55][56][57]. In turn, Aurora kinase Bdependent phosphorylation of histone H3 at Ser10 is required for the recruitment of this protein complex [58]. Thus, abnormal expression and activity of these molecules implies an unfavorable prognosis since they drive tumorigenesis and progression.

Relationship between Aurora kinase A polymorphism and aneuploidy
Both Aurora kinase A and Aurora kinase B play key roles in the regulation of cell division. While Aurora kinase A is responsible for the correct separation of centrosomes during mitosis, Aurora kinase B is an essential part of the chromosomal passenger complex (CPC). AurkB is therefore crucial for the correct chromosomal alignment in the equatorial plane as well as the attachment of the mitotic spindle to kinetochores [55].
Aurora kinase A is not only frequently overexpressed in tumor cells, but elevated levels of Aurora kinase A also cause chromosomal instability and thus errors in cytokinesis which ultimately lead to aneuploidy [26,46,59]. Although the molecular mechanisms differ, overexpression of Aurora kinase B is also suspected to cause chromosomal instability [60,61]. Flow cytometric evaluation of the homozygous cell lines used in this study revealed predominantly diploid, and only rarely tetraploid, cells. In contrast, we observed an enormous increase in heterozygous cells in an aneuploid (i.e., tetraploid) state. This nding con rms previous work performed on SAS and UD-SCC-5 cells treated with different inhibitors [62]. Most importantly, the formation of aneuploid cells occurred only in a heterozygous HN cell line (i.e., HN cells).
In addition, the degree of aneuploidy could also be increased by si-RNA mediated knockdown of Aurora kinase B [39].

Correlation of HPV status and Aurora kinase A polymorphism with overall survival
The incidence of HPV-induced HNSCC in the oropharynx has signi cantly increased in recent years, and especially among men, the incidence is raising drastically [9,63,64]. Overall, the prevalence of HPV infection in HNSCC varies from approximately 25-43%, depending on the location and method used to determine HPV status [12,11,[65][66][67]; in this study we identi ed 49.5% HPV samples.
It has been frequently reported that HPV infection not only contributes to the development of tumors, but also represents a favorable prognostic marker for the survival of patients with HNSCC. A positive HPV status correlates with a prolonged overall survival [41,[68][69][70][71][72], which we have con rmed in the present study. In contrast to the impact of HPV infection, high expression of tumor associated Aurora kinase A correlates with a pronounced increase in tumor progression, and consequently a shortened overall survival of the patients [37,51,73,74]. This is in agreement with the ndings reported by Reiter et al. [37].
In addition, clinically relevant associations between the polymorphism of Aurora kinase A and the outcome of disease have been identi ed [31]. For example, Aurora kinase A is suspected to initiate cell migration and invasion and thus increase the metastatic potential of HNSCC [75]. However, the predictive power of the AurkA polymorphism on overall survival remains controversial. Although Royce et al. detected Aurora kinase A overexpression in the tumor tissue, this did not correlate with the prognosis of the patients [76]. Results from our research group con rm this, as there was no detectable association between the Aurora kinase A polymorphism and overall patient survival [62,39]. However, the literature clearly raises suspicion that the Aurora kinase A polymorphism signi cantly increases the risk of developing malignancy [77], and also plays a crucial role in the response to treatment. We previously demonstrated that the Aurora kinase A polymorphism signi cantly correlated with the response to treatment with the EGF receptor antibody cetuximab in vitro. We showed that homozygous HNSCC cells harboring the AurkA Phe/Phe genotype were sensitive to cetuximab treatment; by contrast, clonogenic survival of cells with a heterozygous genotype (Phe/Ile Aurora kinase A) could not be impaired by cetuximab. The inhibition of Aurora kinase A with speci c si-RNA in combination with cetuximab also led to a statistically signi cant reduction of colony formation only in the heterozygous Phe/Ile cell line [39].
In the subsequent work of 2018, the relevance of the Aurora kinase A polymorphism in terms of therapeutic response was con rmed in vitro. Correlation analyzes revealed a signi cantly better response to speci c inhibition therapy in the AurkA heterozygous Phe/Ile cell line [62].
These varying results can be explained by several aspects. In the previous study, the treatment with the monoclonal antibody cetuximab was speci cally investigated, whereas in the later work docetaxel with speci c Aurora kinase inhibitors was used. On the other hand, in 2014 the survival data was derived from a colony formation assay, while in 2018 cell viability was assessed.

Conclusions
In order to estimate the impact of the Aurora kinase A Phe31Ile polymorphism on the outcome of HNSCC disease in greater detail, a prospective study in the palliative setting is needed. Prospectively, the AurkA polymorphism, together with the HPV status, could contribute to an enhanced patient strati cation that might be relevant for therapeutic decision-making. A prerequisite for improved treatment e cacy is an accurate and precise diagnostic determination of the presence of HPV infection and Aurora kinase A Phe31Ile polymorphism.

Declarations
Ethics approval and consent to participate The study with all experimental protocols were approved by the Ethics Committee of the Klinikum rechts der Isar, Technical University Munich (project number 1420/05). All methods were carried out in accordance with relevant guidelines and regulations. Informed consent was obtained from all subjects.

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
MW was a major contributor in study design and helped with data curation.
GB was involved in methodology and in vitro experiments.
WW helped with data curation and provided patient data.
AP was head of project administration and a major contributor in writing the manuscript.
All authors read and approved the nal manuscript.