Effect of neoadjuvant chemotherapy and its correlation with HPV status, EGFR, Her-2-neu, and GADD45 expression in oral squamous cell carcinoma

Background Head and neck cancers are the commonest cancer in Southeast Asia. Despite being a surface cancer, it is associated with significant morbidity as despite early detection by the patients they often report for treatment late and hence are associated with poor prognosis. The role of neoadjuvant chemotherapy in head and neck cancer is still under evaluation; there is a large subgroup of population that does not respond to chemotherapy, and hence, most studies have failed to show any survival benefit. This study evaluated the role of neoadjuvant therapy with docetaxel and carboplatin in patients with oral cancer and correlated the response to human papilloma virus, EGFR1, EGFR2, and GADD45 expression. Methods A total of 24 locally advanced, non-metastatic oral cancer patients were included in the study. Tumor biopsies were taken prior to the start of neoadjuvant therapy for expression of EGFR, Her-2-Neu, and GADD45 by immunohistochemistry and for HPV by PCR. The response was evaluated using Response Evaluation Criteria in Solid Tumors (RECIST) criteria after three cycles of chemotherapy. Statistical analysis was performed using correlation and Kaplan–Meier analysis; the difference in survival was calculated with log rank test. Results A total of 21 male and 3 female with a mean age of 53.12 years were enrolled. Sixty-five percent of these received three cycles of chemotherapy. Five patients were positive for HPV 16 and none for HPV 18. Twenty-two of 24 patients showed GADD45 expression, 3 showed expression of Her-2-Neu while all 24 showed expression for EGFR1 protein. Two-year overall survival was 81%; GADD45 expressions were found to significantly affect the overall and disease-free survival, while any of the other protein expression studied and HPV status was not significant. Conclusion The result of the present study shows significant downgrading of the oral cancers with neoadjuvant chemotherapy suggesting its utility in borderline operable cases. However, the response of chemotherapy does not appear to be related to the expression of EGFR, Her-2-Neu, and GADD45 protein or presence of HPV. Bone involvement, perineural invasion, and GADD45 expression significantly predict OS and DFS. All patients who did not express Gadd45 died before 2 years. Study with more subjects and longer follow-up should be carried out to elucidate this relation further.


Background
Head and neck cancer squamous cell carcinoma (HNSCC) is the sixth most common cancer with an annual incidence of approximately 4,00,000 worldwide. Two third of the global incidence is reported from developing countries, of which one third is from India. The survival rate of HNSCC is poor, and the disease has a poor prognosis. The treatment of early cancer is surgery; however, in later stages, the chemotherapy (CTh) and radiotherapy [1] are preferred. Several new chemotherapeutic agents including taxanes and platinums have been used as first-line treatment in head and neck cancer; however, the response rate with this doublet or triplet combination ranges from 40 to 60%.
Human papilloma virus (HPV) has been reported to be a causative factor in a subset of HNSCC that usually has a favorable prognosis [2]. High-risk HPV (HPV 16, 18, and 33)-related HNSCC is associated with certain sexual behavior, such as oral sex and increasing numbers of sexual partners, and these tumors usually lack association with smoking and alcohol [3]. Several studies and meta-analysis have confirmed that HPV-related HNSCC behaves differently from HPV-negative SCC and is associated with better treatment response to chemotherapy and radiation [4,5].
Growth arrest and DNA damage repair (GADD45) gene plays important and diverse roles in the regulation of cellcycle [6]. Gadd 45 protein interacts with several regulatory pathways [7]. GADD45 promoter hypermethylation is frequently detected in tumor cell lines, including 85% of non-Hodgkin's lymphoma, 50% of Hodgkin lymphoma, 73% of nasopharngeal, 50% of cervical, 29% of esophageal, and 40% of lung carcinomas [7]. The role of GADD45a as a potential therapeutic target has become a matter of interest due to the fact that it is upregulated on docetaxel treatment. Because of the various functions of GADD 45 in cell-cycle control and its dysregulation in cancer, chemotherapeutic and molecular agents targeting GADD45 proteins may serve as novel therapeutic interventions in cancer treatment. Ramachandran et al. [8] described GADD45a novel potential therapeutic target which has been highlighted by the fact that it is upregulated on docetaxel treatment and may contribute to docetaxel mediated cytotoxicity of prostate cancer. In other study, authors found an increase in the GADD45A and GADD45B in docetaxel-and estramustine-treated prostate cancer cells which suggest their affects in the induction of apoptosis [9].
The big challenge is to identify which treatment is best suited for the individual patient by studying tumor biology at a molecular level and identifying biomarkers for predicting the response to therapy. Aim of the study was to study the effect of neoadjuvant chemotherapy with docetaxel and carboplatin in locally advanced non-metaplastic oral squamous cell carcinomas and to correlate the tumor response and survival with HPV status, EGFR, Her-2-neu, and GADD45 expression.

Patients
A total of 24 patients attending the surgical oncology outpatient between December 2009 to August 2011 with histologically proven locally advanced, borderline operable oral squamous cell cancers (OSCC) were included in the study. Those who had a history of severe hypersensitivity reaction to docetaxel were excluded. The study was approved by the Institute Ethics Committee. Informed consent was taken from all the patients prior to recruitment and collection of specimen. Tissue was stored at − 80°C till analysis.
Biopsy was taken to study the HPV positivity, EGFR, GADD45, and Her-2-neu expression. All patients underwent neoadjuvant chemotherapy (NACT) with docetaxel 80 mg/m 2 and carboplatin 300 mg/m 2 on day 1 as a three weekly regimen, and response of therapy was correlated to HPV positivity, EGFR, GADD45, and Her-2neu expression. Responses were evaluated by clinical examination and post therapy CT scan by Response Evaluation Criteria in Solid Tumors (RECIST) criteria (complete response, partial response, progressive disease, and stable disease).

Expression analysis by PCR
DNA isolation DNA isolation was carried out by phenol-chloroform-isoamyl alcohol method.

Immunohistochemistry
Immunohistochemistry was performed on formalin-fixed paraffin blocks which cut at a thickness of 2 μm. These sections were deparaffinized in xylene followed by hydration in a graded series of alcohols. Sections were left under running water for 15 min. Endogenous peroxidase activity was blocked by incubation in 3% hydrogen peroxide for 30 min at room temperature. After rinsing in TBS buffer (pH 7.4) for 30 min, the sections were incubated with primary antibody at 4°C overnight. The following primary antibodies were used: EGFR (SantaCruz Biotechnology, Santa Cruz, CA), GADD45A (Abcam, Cambridge, MA), and Her-2-neu (Biogenex). Slides were then washed again in TBS. Secondary antibody was added, and slides were washed again after 30 min. The labeled antigen-antibody complexes were visualized as brown pigments via a standard DAB (Zhongshan Jinqiao Co., Beijing, China) protocol. DAB was added and kept for 10 min until brown color developed. Slides were washed in running water, and counterstaining was done with hematoxylin. Slides were then blotted, dried, and mounted.

Statistical analysis
Correlations were performed by using Pearson's and Spearman's correlation coefficient with two-tailed significance. Survival was compared using Kaplan-Meier method, and the difference in survival was compared by using log rank test. p < 0.05 was considered as statistically significant. Analyses were performed with SPSS software (version 13.0; SPSS Inc., Chicago, IL, USA).

Clinicopathological features
Out of 24 patients enrolled, 21 (87.5%) and 3 (12.5%) were males and females respectively. Mean age of the study population was 53.12 years (range 25-72 years). Eighty-eight percent of the patients had the habit of tobacco and alcohol, and only 2% patient did not have the habit of tobacco consumption in any form. Most of the cases were of carcinoma of buccal mucosa (29%), lower alveolus (25%), and tongue (25%). The most common symptoms among the patients were ulcer (96%) and pain (71%). Other symptoms include dyspahgia (17%), loosening of teeth (17%), bleeding (13%), trismus (13%), and dysarthia (8%). The primary growth was of ulceroproliferative type in 50% patients and of infiltrative type in 50% cases. Eighteen out of 24 patients had extension to the adjacent skin, muscle, or bone either alone or combination of more than one. Majority of the patients had T4 disease (19; 79%) while 13 (54%) had N2 disease (Table 1).
HPV status, GADD-45, EGFR, and Her-2-neu expression PCR for HPV was performed on the tumor samples in all the 24 patients. Out of 24 samples, 5 were found to be positive for HPV16 type whereas HPV18 was not found in any of the cases (Fig. 1). GADD45 expression was shown in 22 (92%) out of 24 patients. All the patients had cytoplasmic expression of GADD45 (Fig. 2). There was only one patient which had additional staining also. Grade I staining was present in 2 (6%), grade II in 15 (63%), and grade III in 5 (21%) patients (Fig. 2). EGFR was expressed in all the cases (100%). EGFR showed grade I staining in 6 (25%), grade II in 8 (33%), and grade III in 10 (42%) patients (Fig. 3). Out of 24 samples, only 3 (12.5%) samples expressed Her-2-neu. The expression was mild in all the 3 cases (Fig. 4). Out of 24 patients, 2 patients received 2 cycles, 15 received 3, 3 patients received 5, and only 1 patient received 6 cycles of neoadjuvant chemotherapy with docetaxel (80 mg/m 2 ) and carboplatin (300 mg/m 2 ). The response to CTh was assessed according to RECIST criteria. One (4%), 11 (46%), 8 (33%), and 4 (17%) patients had complete, partial, stable, and progressive response on CTh respectively. The overall response rate was 50%. The response of NACT on the size of tumor was compared before and after CTh. The mean length and breadth of the lesions before CTh were 4.18 and 2.8 cm while post CTh, it was 3.22 and 2.6 cm respectively.

Correlation between markers, T and N stage, performance status, and response
Her-2-neu was expressed in tumors with high EGFR expression. The correlation coefficient (r) was 0.4, and p value is 0.048. There was no statistically significant correlation between CTh response and EGFR, Her-2-neu, and GADD45 expression, and HPV status ( Table 2). Patients with HPV-positive tumors have a better performance status (r = + 0.39, p < 0.05). Patients with better performance status have a better response to CTh (r = + 0.52, p < 0.05). No significant correlation was observed between T, N, and composite stage and the markers under study ( Table 2).

Two-year survival
The 2-year overall survival (OS) was 81%. There were 2 females on follow-up, and both of them died within 1 year. In males, the 2-year overall survival was 89% (p = 0.00). Two-year overall survival by GADD45 expression grade was significantly different. Of the 24 patients, only 2 did not express GADD45 and both of them died at 2 years. There was no statistical significant difference in 2-year overall survival with relation to HPV status, EGFR, and Her-2-neu expression (Table 3).

Disease-free survival
The overall 2-year disease-free survival (DFS) in the study was 66.7% (Figs. 5 and 6). Those cases which did not express GADD45 had a 0% 2-year DFS while it was 100, 58.3, and 100% in grade 1, 2, and 3 staining respectively. The difference was statistically significant (p = 0.012); however, it was not consistent pattern. The 2-year DFS in HPV-positive patients was 100% and in HPV-negative patients, 56.2% (p = 0.077); the HPV-positive patients had better DFS; however, it did not reach statistical significance   (Fig. 7). EGFR and Her-2-neu did not correlate significantly with 2-year DFS.

Discussion
The treatment of squamous cell carcinoma of head and neck (HNSCC) has been the realm of surgeons and radiation oncologists. There has been a paradigm shift in the management of HNSCC over the last two decades, and now, chemotherapy (CTh) is an integral part of the management. Therefore, it is a big challenge to individualize the treatment. Thus, this study was done to identify predictors of response to CTh with docetaxelbased regimen in locally advanced oral squamous cell carcinoma and correlated the tumor response with HPV status, EGFR, Her-2-neu, and GADD45 expression. In our study, 46% of patients had partial response to CTh. The complete response rate (CRR) was 41.6%, and overall response rate (ORR) was 50%. Lip tumors responded best to CTh followed by GB sulcus tumors, 57% of buccal mucosa, 50% each of tongue and alveolus lesion responded to CTh. Colevas et al. reported a CRR of 61% and ORR of 100% using the TPFLS regimen in 23 patients with HNSCCC [13]. Their other study also reported a CRR of 63% and ORR of 93% in 30 [15] reported a CRR of 59% and ORR of 88% in 34 patients with HNSCC using the TPF regimen with a lower done of docetaxel. In the phase III TAX323 trial which used induction CT with TPF regimen followed by RT, the CRR was 8.5% and ORR was 68% [16,17]. Albers et al. [18] recently reported an overall response rate of 86% and 3-year survival of 65% using the docetaxel-based chemotherapy in combination in locally advanced cancer wherein 30/45 of his patients were treated with curative intent. Though role of neoadjuvant chemotherapy in head and neck cancers is debatable, however, recent evidence suggests some possible role. Inhestern et al. [19] reported a 97% 2-year OS for responder compared to 76% in those who did not respond to TPF therapy. In all of the patients, the chemotherapy was followed by surgery and concurrent chemoradiation. However, all other studies have failed to show any improvement in the overall or disease-free survival, and some have even showed disease progression and operative complications in patients receiving neoadjuvant chemotherapy [20][21][22][23].  In our study, all the cases expressed EGFR while Her-2neu was expressed in 3 (12.5%) out of 24 patients. Her-2-neu expression was associated with high EGFR expression (correlation coefficient r = 0.4; p = 0.048). Neither EGFR expression nor Her-2-neu expression was significantly associated with response to CTh with docetaxel (r = 0.321; p = 0.126 and r = 0.378; p = 0.69 for EGFR and Her-2-neu respectively) and with 2-year disease-free and overall survival. In an earlier study, EGFR was expressed in 79.4% of cases of recurrent HNSCC [24]. EGFR-positive tumors had a 3-year cause-specific survival of 27.2% after salvage surgery whereas EGFR-negative tumors had a 3-year cause-specific survival of 64.3% (p < 0.001) [24]. The EGFR expression alone had no significant impact on survival [24]. In another study by Shiraki et al., the overall EGFR expression was 39% and in T3 and T4 lesions, it was 37%. In the same study, co-expression of EGFR, p53, and cyclin D1 was associated with shortened survival [11]. Xia et al. observed that at the combine expression of EGFR, Her-2-neu and Her-3 is a stronger predictor of outcome in oral SCC than any individual family members of the EGFR family [12]. EGFR expression (87.5%) was not found to be associated with proliferation, apoptosis, angiogenesis, and lymphangiogenesis in oral SCC [25]. Chen et al. looked at the EGFR and Her-2-neu expression in oral SCC by enzyme immunoassay method. In their study, EGFR was overexpressed in 58% tumors and Her-2-neu in 41%. EGFR overexpression was significantly associated with TNM stage, lymph node involvement and extra capsular spread, and poor survival. Her-2-neu was not found to be associated with any of the above parameters [26].
GADD45 was expressed in 91.6% cases in the study. There was no significant association established between GADD45 expression and response to CTh with docetaxel and carboplatin (r = 0.263; p = 0.236). But a significant association was there between GADD45 expression and 2-year disease-free and 2-year overall survival. Those cases which did not express GADD45 had a poor 2-year overall survival of 0% compared to 81% when compared with positive cases (p = 0.005). There is no published report examining the role of GADD45 as a predictive marker of CT response or survival in LA HNSCC till date. In prostate cancer, upregulation of GADD45 levels has been associated with increased sensitivity to docetaxel in in vitro studies [8]. They have proposed that GADD45 was epigenetically repressed in prostate cancer and its upregulation may be a potential target for therapeutic strategies [8]. Another experimental study had concluded that tumors in GADD45 deficient mice show increased mutation frequency and increased susceptibility to ionizing radiation and to chemical carcinogens [27]. All our cases that expressed GADD45 did so before starting the CTh, and post therapy evaluation was not carried out in this study.

HPV positivity and correlation with CTh response and survival
In our study, PCR for HPV 16 and 18 types were performed and only 5 (20.83%) was found to be HPV 16 type positive. Like our previous study, this study too failed to demonstrate any significant association between HPV status and response to NACT with docetaxel and carboplatin (r = 0.103; p = 0.633) [5]. The 2-year overall survival in HPV-positive patient was 100% and in HPV negative patients, 75%, and the difference was not significant (p = 0.234). The 2-year DFS in HPV-positive patients was 100% and in HPV negative patients were 56.2%, and the difference was almost approached statistically significant (p = 0.077).
The impact of HPV on cancer risk and overall survival in head and neck squamous cell carcinomas has been summarized in a meta-analysis, concluding that 21.95% of HNSCC patients are HPV positive and the prevalent genotype is HPV-16 (86.69%). A better survival in HPVpositive patients has been reported compared to HPVnegative cases [27]. Prevalence of HPV is reported to be 41% in oropharengeal cancers [28]. Fakhry et al. looked  at the impact of HPV positivity on survival in stage 3 and 4 oropharengeal cancers and reported that the HPV-positive patients had higher response rate after induction CTh (p = 0.01) than HPV-negative patients and after chemoradiation treatment (p = 0.007) [28].
Another study also reported that presence of HPV was significantly correlated with a better survival in patients with oral squamous cell carcinoma [29]. Most of the studies have reported a role of HPV as a predictor to response to treatment though our study failed to do so. That may be due to small sample size. Studies with larger sample size may clarify their association better.

Relation of survival with other parameters
In our study, 2-year OS and DFS were 81 and 66.7% respectively. Colevas et al. had got an 83% 2year OS by using TPF regimen [30]. Some other authors also observed a 60% 2-year OS by using TPF regimen [31]. Others have also found a 2-year OS as 79, 60, 41, and 93% [15,[31][32][33]. Another group demonstrated a 3-year OS with the TPF regimen and got only 29.6 [34]. These results showed that the response rates have been variable. It appears that the genetic and molecular characteristics of the tumor do play a role; however, so far, no such

Conclusions
Our findings suggests that HPV status, EGFR, and GADD45 be assessed in OSCC as together they may help in predicting the survival of the patients; though GADD45 expression was found to predict survival and DFS, however, it did not predict the response to chemotherapy with docetaxel.

Acknowledgements None
Funding None

Availability of data and materials
The data is available with the authors.
Authors' contributions MP contributed to the concept and design, data analysis interpretation, and editing of the final manuscript. KKK contributed to the concept and design and data collection analysis and interpretation. RD contributed to the molecular analysis, interpretation of data, and preparation of the draft manuscript. MK contributed to the pathological and IHC analysis and interpretation and preparation of the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate The study was approved by the Institute Ethics Committee, and informed consent was obtained from each participant.

Consent for publication
The written informed consent was obtained from each patient enrolled in this study; the study was approved by the Institute Ethics Committee.