PTEN expression in oral squamous cell carcinoma treated with cetuximab-based chemotherapy

Accepted Manuscript Predictive value of pAKT/PTEN expression in oral squamous cell carcinoma treated with Cetuximab-based chemotherapy Jiong Lyu, M.S, Hao Song, M.S, Zhen Tian, Ph.D, Yuwen Miao, MS, Guoxin Ren, Ph.D, Wei Guo, Ph.D PII:

S2212-4403(15)01186-4

DOI:

10.1016/j.oooo.2015.09.002

Reference:

OOOO 1295

To appear in:

Oral Surgery, Oral Medicine, Oral Pathology and Oral Radiology

Received Date: 22 December 2014 Revised Date:

27 August 2015

Accepted Date: 1 September 2015

Please cite this article as: Lyu J, Song H, Tian Z, Miao Y, Ren G, Guo W, Predictive value of pAKT/ PTEN expression in oral squamous cell carcinoma treated with Cetuximab-based chemotherapy, Oral Surgery, Oral Medicine, Oral Pathology and Oral Radiology (2015), doi: 10.1016/j.oooo.2015.09.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Title page Predictive value of pAKT/PTEN expression in oral squamous cell carcinoma treated with Cetuximab-based chemotherapy

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Authors 1. Jiong Lyu (first author) M.S Department of oral and maxillofacial-head and neck oncology, Ninth people's hospital, Shanghai Jiaotong university school of medicine Shanghai, China 2. Hao Song(co-first author) M.S Department of oral and maxillofacial-head and neck oncology, Ninth people's hospital, Shanghai Jiaotong university school of medicine Shanghai, China 3. Zhen Tian Ph.D Department of oral pathology, Ninth people's hospital, Shanghai Jiaotong university school of medicine Shanghai, China 4. Yuwen Miao MS Department of head and neck surgery, Sir Run Run Shao hospital, Zhejiang University school of medicine Hangzhou China 5. Guoxin Ren Ph.D Department of oral and maxillofacial-head and neck oncology, Ninth people's hospital, Shanghai Jiaotong university school of medicine Shanghai, China 6. Wei Guo (corresponding author) Ph.D Department of oral and maxillofacial-head and neck oncology, Ninth people's hospital, Shanghai Jiaotong university school of medicine Shanghai, China E-mail: [email protected] OR [email protected] Postal address: No. 639 Zhizaoju Rd, Shanghai 200011, China Telephone number: +8621 5039 8070 Fax number: +8621 5090 5789

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Word count of abstract: 116 Word count for complete manuscript: 1915 Number of references: 18 Number of Tables: 2 Number of figures: 4 No virtual microscope images were submitted

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Key words: EGFR inhibitor, Cetuximab, Oral Neoplasm, PTEN, pAKT, Immunohistochemistry

Acknowledgement This work was supported by the Project of Science and Technology Commission of Shanghai Municipality (grants 10410711200 and 08140902100) The authors have no conflict of interest.

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Abstract: Objective: Molecular alterations in downstream effectors of EGFR may confer resistance to EGFR inhibitors. Our aim is to investigate whether PTEN/pAKT expression predict response to cetuximab based chemotherapy in oral squamous cell carcinoma (OSCC). Study Design: We analyzed a cohort of 50 OSCC treated with cetuximab-based induction chemotherapy. The expression of PTEN and pAKT was assessed by immunohistochemistry and their correlation with treatment outcome was analyzed. Results: 18.4% of patients had low PTEN expression and 38.8% had high pAKT expression. Lower pAKT expression were associated with pathological remission (p=0.034) and better disease-free survival (p=0.031). Conclusion: Our study demonstrates that pAKT expression is a predictive biomarker of cetuximab based induction chemotherapy in OSCC.

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Clinical Relevance: Researching biomarkers to predict the efficacy of EGFR inhibitor is indispensable for determining patient specific treatment in OSCC. Our findings showed pAKT expression was a predictive biomarker of response to cetuximab based induction chemotherapy.

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1.Introduction Head and neck cancer, 90% of which is squamous cell carcinoma (HNSCC), is the sixth most common malignancy in the world. Over half of HNSCC present with locally advanced disease and need multimodality therapy with surgery, radiotherapy, chemotherapy and molecular therapy. In recent years, targeted therapy against the epidermal growth factor receptor (EGFR) has become the most promising molecular therapeutics for HNSCC. EGFR plays an important role HNSCC pathogenesis. It can initiate important signal transduction pathways in carcinogenesis. EGFR mRNA and protein were found to be over-expressed in 90% and 40% of HNSCC respectively, and high EGFR expression was associated with poor prognosis.1-3 Though anti-EGFR agents have shown to be promising in the clinical setting, there is one main challenge, namely drug resistance (primary resistance). As monotherapy, Cetuximab (an FDA approved anti-EGFR monoclonal antibody) has a response rate of 15%.2 Thus, identifying biomarkers that can be used to predict response to anti-EGFR therapy is important. Several mechanisms that may contribute to EGFR inhibitor resistance have been summarized in recent reviews.2, 4, 5 One of them is molecular alterations in EGFR downstream effectors, e.g. abnormality in RAS/RAF/MAPK pathway.6, 7. In addition to RAS/RAF/MAPK pathway, EGFR can also activate another critical downstream pathway—PI3K/AKT pathway, which regulates cell growth, survival, motility and metabolism in both normal physiology and cancer.8 Alteration in PI3K/AKT pathway may also have an impact on anti-EGFR efficacy. PTEN and AKT are two important components of PI3K/AKT pathway.(Figure 1) PTEN is a tumor suppressor which antagonizes PI3K function by dephosphorylating PIP3 to regenerate PIP2. Loss of PTEN expression is observed in ~30% of HNSCC.9, 10 AKT is a serine/threonine protein kinase and can be activated by PIP3. Activated AKT (pAKT) has multiple downstream targets involved in cell survival, growth, proliferation, angiogenesis, metabolism and migration. It is reported that pAKT often over-expressed in HNSCC. Whether alteration of PI3K/pAKT expression predicts anti-EGFR efficacy is still unknown. In this study, our aim was to investigate the association between response to Cetuximab-based chemotherapy and PTEN/pAKT expression in oral squamous cell carcinoma (OSCC). We analyzed a cohort of 50 OSCC who underwent cetuximab based induction chemotherapy. Expression of PTEN and pAKT was analyzed by immunohistochemistry and their correlation with treatment outcome was investigated. 2.Materials and Methods Patients This study was performed in accordance with the Declaration of Helsinki, and was approved by the Ethics Committee of Shanghai Ninth People’s Hospital. Fifty OSCC with available biopsy tissue samples who underwent cetuximab-based induction chemotherapy followed by locoregional treatment in our department between September 2011 and April 2013 were evaluated. Clinical and pathological records were reviewed retrospectively. The following data were collected: age, gender, smoking and alcohol history, tumor staging, histological grade, treatment information, pathological results, relapse and survival. The primary endpoint was pathological remission, and the secondary endpoint were overall survival (OS) and disease free survival (DFS). Pathological remission was defined as disappearance of invasive cancer cells in the primary tumor. OS was

ACCEPTED MANUSCRIPT measured from the date of initial diagnosis to death due to any cause. DFS was measured from the date of initial diagnosis to relapse/metastasis.

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Immunohistochemical staining All samples were obtained from diagnostic biopsy before cetuximab-based chemotherapy, fixed in 10% formalin and embedded in paraffin. The tissues were cut into 4 um serial sections and then deparaffinized using xylene and rehydrated through an ethanol series to water. After deparaffinization, endogenous peroxidase blocking and heat-induced epitope retrieval, primary rabbit polyclonal antibody to PTEN (10047-1-AP, Proteintech Group Inc) and primary rabbit monoclonal antibody to pAKT (pS473, Epitomics) at 1:100 dilution were added for treatment

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overnight at 4℃. Then immunohistochemical staining was performed using DAKO Real Envision Detection System following the procedure recommended by the manufacturer. Substitution of the primary antibody with PBS was performed as negative control. Primary lung cancer with a strong staining pattern of PTEN or pAKT was used as a positive control. The positive staining for PTEN was defined as cytoplasmic staining. Positive staining for pAkt was defined as cytoplasmic staining.11

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Evaluation of immunostaining Immunohistochemical staining of pAKT and PTEN was recorded considering both the staining intensity and the proportion of positive tumor cells. Intensity was graded as follows: 0, no staining; 1, weak staining; 2, moderate staining; 3, strong staining. Area was graded as follows: 0, 0-5%; 1, 5-15%; 2, 15-30%; 3, 30-50%; 4, >50%. Staining index = staining intensity multiplied by positive area. The expression index was assessed by two pathologists (L. W. and Z. T.) who were blinded to clinical features. High expression of pAKT was defined as staining index ≥6. Reduced PTEN expression was defined as staining index <9. The cut-off point of pAKT/PTEN were based on the distribution of staining results.

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Statistical Analysis Descriptive statistics were used to describe patients' pathological and clinical characteristics. Chi-square test was used to test the correlation of clinicopathologic variables with expression of PTEN and pAKT. OS and DFS were analyzed by Kaplan-Meier method and log-rank test was performed to test difference in survival between subgroups. Multivariate analysis was performed using the Cox regression model. All tests were two-sided. All statistical analyses were done by SPSS program (version 13). 3.Result The clinicopathological characteristics of 50 OSCC are summarized in Table 1. None of them had a previous history of radiotherapy, chemotherapy, targeted therapy or surgery (except biopsy). Before locoregional treatment, all patients received induction chemotherapy for 6 weeks, which consisted of cetuximab (400 mg/m2 on day 1 and 250 mg/m2 weekly on days 1, 8, 15, 22, 29 and 36), docetaxel (75 mg/m2 intravenously on days 1 and 22) and cisplatin (75 mg/m2 on days 1 and 22). The overall response rate was 66.0%. After induction chemotherapy, 45 patients received surgery with/without post-operative radiotherapy and 5 patients received radical radiation therapy. In 45 patients who received surgery, 22.2% showed pathological remission in primary

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Immuno-histochemical staining and correlation analysis Immunostaining evaluation was successfully performed on 49 specimens except one that did not contain adequate amount of cancer tissue. Forty (81.6%) patients had high PTEN expression while 9 (18.4%) patients had low PTEN expression. Nineteen (38.8%) patients had high pAKT expression while 30 (61.2%) had low pAKT expression (Figure 2). Then we analyzed the correlation of PTEN and pAKT expression with clinicopathologic characteristics including age, gender, smoking, alcohol use, clinical T stage, clinical N stage and WHO histological differentiation (Table 2). Chi-square test showed that poorly differentiated tumors expressed lower PTEN levels compared to both well and moderately differentiated categories (p=0.034). To determine whether the expression level of PTEN and pAKT was associated with the response to cetuximab-based chemotherapy, we analyzed the PTEN and pAKT expression based on pathological remission (Table 2). Chi-square test showed that patients with pathological remission had lower pAKT expression (p=0.034).

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Survival outcome The median follow-up was 28 months. Two-year DFS rate in patients with low expression of PTEN was 53.3% compared with 71.9% in patients with high expression of PTEN (p=0.329, Figure 3A). Two-year OS rate in patients with low expression of PTEN was 44.4% compared with 71.7% in patients with high expression of PTEN (p=0.057, Figure 3B). Two-year DFS rate in patients with low expression of pAKT was 79.9% compared with 49.9% in patients with high expression of pAKT (p=0.031, Figure 4A). Two-year OS rate in patients with low expression of pAKT was 75.2% compared with 52.5% in patients with high expression of pAKT (p=0.132, Figure 4B). In Cox regression model involving gender, age, smoking, alcohol use, tumor stage and pAKT expression, pAKT was an independent prognostic factor for DFS.(p=0.014, HR=0.245 (Low vs. High))

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4.Discussion Primary resistance to anti-EGFR agents is a relevant issue in advanced OSCC. Researching biomarkers to predict anti-EGFR therapy efficacy is indispensable for determining patient specific treatment.5 PI3K/AKT is an important EGFR downstream oncogenic pathway and often altered in OSCC. The present study aimed to investigate whether PTEN/pAKT expression was associated with response to Cetuximab-based induction chemotherapy in OSCC. In our clinical analysis using immunohistochemistry, we found that high expression of pAKT was associated with worse outcome. In our immunohistochemistry analysis, we found that PTEN expression was correlated with histological differentiation. This result is consistent with previous studies.10 However, PTEN expression was not correlated with outcome to cetuximab-based therapy. A previous in vitro study showed that PTEN silencing in HNSCC cell did not lead to loss of cetuximab efficacy in inhibiting EGFR-downstream signaling pathways.12 So we conclude PTEN loss may be not a predictive biomarker of cetuximab. Our immunohistochemistry results showed that pAKT expression level was associated with

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pathological remission and DFS. Several previous researches in colon/lung cancer have reported that pAKT expression predicted EGFR inhibitor efficacy: in colorectal cancer receiving irinotecan and cetuximab, pAKT expression was independently correlated with response rate and outcome13. In non-small cell lung cancer, it was reported that EGFR mutation and pAKT expression predicted gefitinib efficacy.14 In this study, we find that pAKT is a predicting biomarker, but what caused AKT hyper-activation is unclear. According to previous knowledge, activation of AKT can occur not only via EGFR over-expression itself, but also through genetic alterations of PIK3CA or PTEN4. Moreover, abnormally activated pathways which have crosstalk with EGFR pathway may also cause AKT hyper-activation. For example, HER2 amplification, hyper activated IGF-1R signaling pathway and MET mutation/amplification can all result in persistent activation of the PI3K/AKT signaling pathway.15-17 In most situations, even upon EGFR blockage, the PI3K/AKT pathway can remain active and contribute to resistance to EGFR targeted therapy. We think in these situations, addition of AKT inhibitor may improve efficacy of anti-EGFR therapy.

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Our study has several limitations. First, the patients in our study received not only cetuximab but also cytotoxic agents. In breast cancer, it was reported that pAKT expression was related to clinical response to chemotherapy18. Therefore, in our conclusion, the existence of cytotoxic agents is a confounding factor (though whether pAKT predicts response to chemotherapy in OSCC is still unknown). Further study is needed to research the predictive role of pAKT expression in OSCC treated with EGFR inhibitor as monotherapy. Second, the patients who received cetuximab-based induction chemotherapy in our study did not have prior treatment. Thus the predictive value of pAKT expression in relapsed/metastatic OSCC should be investigated in further studies. Third, in this study we used p-AKT ser473 antibody, a widely used marker for Akt activity. However whether p-AKT thr308 (another phosphorylation site) antibody also have predictive value needs further study. Fourth, a small part of patients (10%) received only radiation therapy, which might have an impact on DFS and OS. Fifth, in this study the number of poorly differentiated tumors is small. Studies with a larger sample size would be beneficial to interpret the correlation between PTEN expression and histological differentiation. Moreover, though we find high pAKT expression is related to worse outcome, we do not known which factor causes AKT hyper-activation. Since mutation, copy number alteration, epigenetic change and post-transcriptional control in numerous genes may all contribute to AKT activation, systemic genomic analysis is necessary to clarify this issue. In conclusion, our study demonstrates that in OSCC patients, pAKT expression is a predictive biomarker of response to cetuximab based induction chemotherapy. We conclude that further efforts are needed to research if combined use of AKT/mTOR inhibitors may help to increase the efficacy of EGFR inhibitors in patients with hyper-activated AKT.

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Figures Figure 1 Schematic diagram of PI3K/AKT pathway

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Figure 2. Representative examples for immunohistochemical staining of PTEN and pAKT. A: H&E stain; B1: PTEN low expression; B2: PTEN moderate expression B3: PTEN strong expression; C1: pAKT no expression;C2: pAKT low expression;C3: pAKT moderate expression;C4: pAKT strong expression.(all images were 400X).

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Figure 3. Disease free survival (A) and overall survival (B) by expression level of PTEN. (p=0.329 and 0.057)

Patients' characteristics The correlation of clinicopathologic variables with expression of PTEN and pAKT

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Tables Table 1 Table 2

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Figure 4. Disease free survival (A) and overall survival (B) by expression level of pAKT. (p=0.031 and 0.132)

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No.

%

Total Sex Male Female Age Range (median) <60 ≥60 Site Tongue Buccal Gingiva Floor of mouth Palate T stage T2 T3 T4 N stage N0 N1 N2 Stage III IVa Pathological differentiation Well Moderate Poor Tobacco history Current/former Never Alcohol History Current/former Never

50

100

37 13

74.0 26.0

35-73 (58) 30 60.0 20 40.0 58.0 4.0 18.0 14.0 6.0

7 7 36

14.0 14.0 72.0

12 20 18

24.0 40.0 36.0

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29 2 9 7 3

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8 42

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Patients' characteristics

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Table 1

16.0 84.0

15 30 5

30.0 60.0 10.0

33 17

66.0 34.0

26 24

52.0 48.0

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≥60

Gender (n=49) Clinical T stage (n=49)

Clinical N stage (n=49)

Differentiation (n=49) Smoking (n=49) Alcohol use (n=49) Pathological remission (n=44)

Male Female T2 T3 T4 N0 N1 N2 Well Moderate Poor Yes No Yes No Yes No

Low

High

P

Low

High

P

29 20 37 12 7 6 36 11 20 18 14 30 5 33 16 26 23 10 34

6 3 5 4 1 1 7 2 3 4 3 3 3 4 5 3 6 2 6

23 17 32 8 6 5 29 9 17 14 11 27 2 29 11 23 17 8 28

0.613

19 11 21 9 5 4 21 8 11 11 9 18 3 20 10 16 14 9 18

10 9 16 3 2 2 15 3 9 7 5 12 2 13 6 10 9 1 16

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*. Correlation is significant at the 0.05 level (2-side)

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0.123 0.943

0.848

0.026*

0.105 0.189

0.865

0.260 0.775

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<60

Patients

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Age (n=49)

pAKT expression

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Variable

PTEN expression

0.625

0.962

0.898 0.962 0.034*

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Clinical Relevance: Researching biomarkers to predict the efficacy of EGFR inhibitor is indispensable for determining patient specific treatment in OSCC. Our finding showed pAKT expression is a predictive biomarker of response to cetuximab based chemotherapy.