Biomarkers in diagnosis and therapy of oral squamous cell carcinoma: A review of the literature

Accepted Manuscript Biomarkers in diagnosis and therapy of oral squamous cell carcinoma: A review of the literature Sebastian Blatt, DMD, Maximilian Krüger, MD, DMD, Thomas Ziebart, MD, DMD, PhD, Keyvan Sagheb, MD, DMD, Eik Schiegnitz, MD, DMD, Elisabeth Goetze, Bilal Al-Nawas, MD, DMD, PhD, Andreas Max Pabst, MD, DMD PII:

S1010-5182(17)30055-0

DOI:

10.1016/j.jcms.2017.01.033

Reference:

YJCMS 2596

To appear in:

Journal of Cranio-Maxillo-Facial Surgery

Received Date: 2 December 2016 Revised Date:

22 December 2016

Accepted Date: 30 January 2017

Please cite this article as: Blatt S, Krüger M, Ziebart T, Sagheb K, Schiegnitz E, Goetze E, Al-Nawas B, Pabst AM, Biomarkers in diagnosis and therapy of oral squamous cell carcinoma: A review of the literature, Journal of Cranio-Maxillofacial Surgery (2017), doi: 10.1016/j.jcms.2017.01.033. 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 Biomarkers in diagnosis and therapy of oral squamous cell carcinoma: A review of the literature

Sebastian Blatt (DMD) 1; Maximilian Krüger (MD, DMD) 1; Thomas Ziebart (MD, DMD,

RI PT

PhD) 3; Keyvan Sagheb (MD, DMD) 1; Eik Schiegnitz (MD, DMD) 1; Elisabeth Goetze; Bilal Al-Nawas (MD, DMD, PhD) 1; Andreas Max Pabst (MD, DMD) 2, 1

Department of Oral- and Maxillofacial Surgery, University Medical Center, Augustusplatz

SC

1

2, 55131 Mainz, Germany (Head: Univ.-Prof. Dr. Dr. W. Wagner) 2

Department of Oral- and Maxillofacial Surgery, Federal Armed Forces Hospital,

3

M AN U

Rübenacherstrasse 170, 56072 Koblenz, Germany (Head: Prof. Dr. Dr. R. Werkmeister) Department of Oral- and Maxillofacial Surgery, University Hospital Giessen and Marburg,

Campus Marburg, Baldingerstrasse, 35043 Marburg, Germany

Sebastian Blatt, DMD

TE D

Corresponding author

Department of Oral- and Maxillofacial Surgery University Medical Center

55131 Mainz

AC C

Germany

EP

Augustusplatz 2

Phone: +49/6131/173761 Fax: +49/6131/176602

Email: [email protected]

Sources of support There are no sources of support.

ACCEPTED MANUSCRIPT Summary Oral squamous cell carcinoma (OSCC) represents the sixth most common cancer, accounting for 2-4% of all malignancies worldwide. The overall survival rate of less than 60% remains

RI PT

generally poor, with prognosis heavily relying on the TNM staging system. Tumor size as well as the presence and extent of lymph node metastases are widely recognized as the most important predictors. However, the underlying mechanisms that lead to an aggressive

phenotype are not yet fully understood. Therefore, possible biomarkers are much in need to

SC

predict prognosis, to help individualize therapy approaches, and to overcome possible

M AN U

resistance mechanisms.

Despite a multitude of recently published biomarkers for OSCC, there is still an ongoing debate regarding their implementation in the clinical workflow. Thus, a systematic literature search via PubMed was performed to update the current literature with the latest evidence. In total, 128 studies were included and over 100 different biomarkers evaluated with reference to

TE D

their influence of survival, tumor recurrence, advanced grading and lymph node metastasis. In this review, we highlight the important molecular mechanism underlying possible markers

EP

in tissue, blood or saliva samples for OSCC. As a major result, no clinical trials could be obtained to prove clinical importance of the validated predictors for survival, tumor

AC C

recurrence, lymph node metastasis and therapy resistance. Therefore, further clinical investigations are much needed.

Keywords: oral squamous cell carcinoma, oral cancer, OSCC, biomarker, diagnosis, therapy

ACCEPTED MANUSCRIPT INTRODUCTION Oral squamous cell carcinoma (OSCC) accounts for 95% of all oral cavity and oropharyngeal cancers and represents the sixth most common cancer, accounting worldwide with

RI PT

approximately 300,000 new cases annually (Nagata et al., 2015; Schiegnitz et al., 2012; Skrinjar et al., 2015). The main risk factors for developing oral cancer are smoking and heavy alcohol consumption, especially a combination of both (Blatt et al., 2016b; Brockmeyer et al., 2014; Kruger et al., 2015b). Surgical therapy remains the main treatment approach, while

SC

adjuvant therapy such as radiation or chemotherapy may be used in combination with either

M AN U

treatment in advanced cases (Chu et al., 2012). Despite advances in these clinical treatments, overall survival rates of OSCC remain generally poor at approximately 50-60% (Seki et al., 2011; Tang et al., 2013). This is mainly due to frequent late diagnosis in up to 50% of the cases showing lymph node metastasis at the time of initial clinical investigation (Hamada et al., 2012b; Morandi et al., 2015). Apart from tumor size, the presence of lymph node

TE D

metastasis is regarded as the single most important predictor for patient outcome (Huang et al., 2012; Kono et al., 2013; Sagheb et al., 2016). In the initial clinical staging, the TNM

EP

classification is used, but it appears to be insufficient to accurately predict tumor aggressiveness or to select treatment modalities on an individual basis. Patients with the same

AC C

TNM stage may have different clinical behaviors, different treatment responses, and even different outcomes (Fu et al., 2016; Gissi et al., 2016; Oliveira-Costa et al., 2015). Therefore, a more detailed understanding of the molecular events that potentiate growth and dissemination is necessary and can lead to the discovery of novel biomarkers that predict lymph node metastasis, tumor recurrence or overall survival (Huber et al., 2011; Shi et al., 2011). Furthermore, they may predict treatment response and therefore alter therapy options individually (Blatt et al., 2016a; Kruger et al., 2016; Kruger et al., 2015a). A multitude of such lately found predictors have been published (Safi et al., 2015). Aside from “solid”

ACCEPTED MANUSCRIPT biomarkers, invasively measurable in tumor tissue itself, molecular markers that are detectable in body fluids such as blood or saliva may be able to predict the development of OSCC at an even earlier or precancerous stage, combined with the advantages of accessibility, minimally invasive procedures, relatively low cost, and multiple sampling for

RI PT

monitoring (Jiang et al., 2015; Sartini et al., 2012). However, there is still an ongoing debate regarding their clinical importance, as evidence of implementation in the therapeutic/clinical workflow remains sparse.

SC

The aim of this review is to summarize, highlight and evaluate the latest evidence in the

M AN U

recent literature (published in the last 5 years) in order to determine the clinical importance of diverse biomarkers used for prediction of lymph node metastasis, tumor recurrence, radiation resistance and/or overall survival for patients with OSCC .

Consequently, a systematic literature search was performed to compare recently found liquid

TE D

and/or solid biomarkers and their clinically significance for the following: cell cycle regulation, proliferation and apoptosis; cell motility, adhesion and extracellular matrix degradation/microenvironment; and transcription factors, immunologically reactions and

AC C

EP

angiogenesis.

ACCEPTED MANUSCRIPT MATERIALS AND METHODS A Medline literature search was performed via PubMed with the following search narratives: medical subject heading (MeSh) term “oral squamous cell carcinoma” was searched in

RI PT

combination with “biomarker” and “long-term survival”, “local recurrence”, “metastasis” as well as “radiation resistance” and “chemo resistance”. Search results were filtered for

literature between 01/07/2011 and 06/30/2016. The Search was conducted between May and

SC

June 2016. The latest update was 06/30/2016.

As inclusion criteria, we reviewed all clinical trials, in vivo and ex vivo prospective as well as

M AN U

retrospective studies investigating novel solid and/or liquid biomarkers for OSCC and their significance on tumor recurrence, lymph node metastasis, radiation/chemo resistance and long-term survival. Furthermore, investigated parameters were sample type (tumor tissue vs. saliva/blood sample), molecular mechanism (over- vs. underexpression), study population and

TE D

p-value for the respective biomarker. As exclusion criteria, we determined studies that analyzed other tumor types than OSCC, in vitro studies only, a study population <30, a p-

AC C

EP

value >0.05, reviews and studies in languages other than English.

ACCEPTED MANUSCRIPT RESULTS Overall, the search narrative resulted in 1,968 hits. After reading the title and/or abstract, first 39 duplicates were excluded. A total of 128 studies then met the inclusion criteria, and full-

RI PT

text was obtained for further analysis. Altogether, more than 100 different molecular biomarkers were analyzed referring to their significant correlation with lymph node

metastasis, tumor recurrence, radiation resistance and/or long-term survival as stated in the study. A total of 44 studies were identified that analyzed 43 different molecular types of

SC

biomarkers concerning cell cycle regulation, proliferation and apoptosis (Table 1). As sample

M AN U

type, all of the investigated markers were “solid marker” invasively obtained from tumor tissue. Three reports verified the in vivo results additionally in cell lines (Ota et al., 2014; Xie et al., 2014; Yang et al., 2014). Regarding cell motility, adhesion and extracellular matrix degradation/microenvironment, 32 studies were obtained analyzing 29 different types of biomarkers (Table 2). Among them, five studies showed additionally in vitro analysis (Goto

TE D

et al., 2014; Grimm et al., 2012; Li et al., 2014; Yoshizawa et al., 2013). Five studies evaluated possible liquid biomarkers, all obtained from patients’ blood samples (Ding et al.,

EP

2014; Hsin et al., 2014; Hsu et al., 2015; Jiang et al., 2015; Lin et al., 2012). For transcription factors, immunologic reactions and angiogenesis, 46 studies were found that investigated 36

AC C

different groups of biomarkers (Table 3). Among them, 10 studies combined in vivo with in vitro analysis (Fang et al., 2014; Koyama et al., 2015; Lin et al., 2015a; Lin et al., 2015b; Sasahira et al., 2014; Shen et al., 2014; Shiiba et al., 2013; Shin et al., 2016; Wu et al., 2012; Yanase et al., 2014). Furthermore, seven studies examined liquid biomarkers (Chen et al., 2014; Cheng et al., 2012b; Kammerer et al., 2013; Skrinjar et al., 2015; Sun et al., 2016; Xu et al., 2016). One study by Sartini et al. was obtained that analyzed level of nicotinamide Nmethyltransferase as a possible biomarker in tumor tissue and saliva in the same cohort (Sartini et al., 2012). Overall, no single trial that showed the use of the respective biomarker

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

in the clinical workflow could be found.

ACCEPTED MANUSCRIPT DISCUSSION Biomarkers for cell cycle regulation, proliferation and apoptosis In general, tumor progression is characterized by an imbalance between cell proliferation and

RI PT

apoptosis (Kato et al., 2011). Here, sustained proliferative signaling is a hallmark of malignant transformation (Hanken et al., 2014; Klimowicz et al., 2012; Liu et al., 2015). There are some molecular features like antigens expressed during the G1, S and G2 phases of

SC

cycling cells but not in the G0 phase (Liu et al., 2015). Therefore, these molecules can be used as potential biomarkers when predicting sustained proliferation. Key players in the cell

M AN U

cycle regulation are cyclins that are regulated by cyclin-dependent kinases (Hanken et al., 2014). One of them is the cyclin D1 gene, a proto-oncogene located on chromosome 11q13, which encodes a positive cell-cycle regulator that promotes cell-cycle progression from the G1 to the S phase (Zhong et al., 2013). In this context, high cyclin-D1 level in tumor tissue

TE D

can be used as a potential biomarker for long-term survival in OSCC (p=0.006, 0.0127 and 0.029 respectively) (Hanken et al., 2014; Huang et al., 2013; Murali et al., 2016). In addition, cyclin D1 underexpression could be used as a predictor for resistance to induced

EP

chemotherapy when patients present with cN2 OSCC (p=0.025) (Zhong et al., 2013). Interestingly, some of the cell cycle regulation molecules are suitable as predictors for

AC C

radiosensitivity, which was shown by Freudlsperger et al for underexpression of antigen Ki67 (p=0.048) (Freudlsperger et al., 2012). Klimowicz et al. demonstrated that a low level of Ki-67 in OSCC tumor tissue is an independent predictor for long-term survival (p<0.006). To increase clinical utility, the authors suggest tumor type and treatment-specific measurement techniques to achieve optimal proliferation measurement standards (Klimowicz et al., 2012). Furthermore, there are several kinases involved in regulating cellular functions such as proliferation that can be analyzed in tumor tissue and used as biomarker for long-term survival such as src protein (p=0.00267), CDC7 (p=0.01) and CDC20 (p=0.032) as well as for

ACCEPTED MANUSCRIPT tumor recurrence like protein kinase C (p=0.016) (Cheng et al., 2013; Cheng et al., 2012a; Chu et al., 2012; Moura et al., 2014). Another important regulator of the cell cycle and therefore for the integrity of the genome is tumor suppressor protein p53. Alteration of the p53 gene is the most commonly reported genetic abnormality in many types of cancer as well

RI PT

as in OSCC (Kato et al., 2011). Other proteins like the transmembrane glycoproteins MUC1 and MUC4 modulate p53 themselves. These mucines can directly bind to the p53 regulatory domain and are involved in cell differentiation, renewal of the epithelium and modulation of

SC

cell adhesion, immune response and cell signaling. As a survival response to stress, the

organism thereby decreases cell death by selectively promoting the transcription of growth

M AN U

arrest genes and decreasing the transcription of apoptotic genes (Hamada et al., 2012a; Hamada et al., 2012b; Kamikawa et al., 2015). Another key inhibitor of tumor suppressor p53 is iASPP (inhibitor of apoptosis-stimulating protein of p53) that is overexpressed in several human cancers. Interestingly, overexpression of iASPP is associated with resistance to

TE D

cisplatin-induced apoptosis and radiation therapy (Kim et al., 2015). Proliferating cell nuclear antigen (PCNA) is a factor in DNA replication that interacts indirectly with p53 over p21 (Kato et al., 2011). Here, the overexpression of p21 and p27 enhances the binding to cyclin-

EP

CDK complexes, which inhibits cell proliferation (Zhang et al., 2013). The retinoblastoma (Rb) pathway, as the common conduit for overcoming the restriction point to S-phase entry,

AC C

has been shown to be abrogated upstream by positive regulators such as cyclin D1 and CDK6 or by negative regulators such as p27, p21, p16 and p19 in OSCC (Murali et al., 2016). Therefore the majority of the above-mentioned molecules were evaluated as potential biomarkers for aggressive tumor growth of OSCC mirrored by the statistically significant correlation with long-term survival as displayed in Table 1. In order to sustain cytosolic homeostasis, cells activate a further set of tightly controlled regulatory programs. For instance, heat shock proteins, called chaperones, help in assembling,

ACCEPTED MANUSCRIPT folding and transporting newly synthesized proteins. One of them (GRP78) also serves as a sensor for cytosolic stress, such as in patients with genetic mutations affecting secretory protein or chemical substances (Xia et al., 2014). Autophagy is another eukaryotic degradative mechanism which maintains cellular homeostasis in environmental stress. The

RI PT

role of autophagic degradation in cancer cells is ambiguous, as it may promote or suppress carcinogenesis. Here, autophagy has been shown to act both as a tumor suppressor and as a mechanism to sustain survival. Microtubule-associated protein light chains 3 (LC3) is a

SC

reliable marker for monitoring autophagy and correlates statistically significantly with longterm survival (p=0.0001) and tumor recurrence (p=0.026) (Tang et al., 2013; Tang et al.,

M AN U

2015). The cytoskeleton and a couple of other transmembrane proteins are involved in maintaining tissue homoeostasis, cell growth control and development (Ma et al., 2016). Connexins are responsible for the gap junctional intercellular communication (GJIC), and their overexpression may serve as a predictive marker for long-term survival of patients with

TE D

OSCC (p=0.0088).

In pathological conditions like cancer, there is a significant imbalance between the production

EP

and removal of reactive oxygen species (ROS), resulting in irreversible oxidative damage to DNA and proteins, interfering with important cellular function (Huang et al., 2013). This

AC C

phenomenon is also exploited in adjuvant therapy when ionizing radiation or alkylating agents induce cytotoxic damage and apoptotic DNA double-strand breaks. As a defense strategy, cancer cells encompass the rapid synthesis and degradation of poly(ADP-ribose) by cellular poly(ADP-ribose) polymerases (PARPs) that physiologically facilitate DNA repair through relaxation of the chromatin structure (Mascolo et al., 2012). Furthermore, metallothioneins (MTs) protect the cell against alkylating agents, oxygen radicals and ionizing radiation through the donation or chelation of elements and redox activity (Brazao-Silva et al., 2015). However, in this study, GDF15 expression (p=0.019) and a low expression of the above-

ACCEPTED MANUSCRIPT mentioned cyclin D1 (p=0.001) were found to serve as predictive biomarkers for a benefit from docetaxel, cisplatin, 5-fluorouracil (TPF)−based induction chemotherapy (Yang et al., 2014). In summary, there are several biomarkers for cell cycle regulation, proliferation and

RI PT

apoptosis. In our review of the literature, we found seven predictors of lymph node

metastasis, four of tumor recurrence and three that were associated with resistance to adjuvant therapies. Although most of these markers, especially p53 and the subgroup of cyclins, are

M AN U

showing the lack of translation into clinical set-up.

SC

well documented in the recent literature, no clinical trial could be obtained in our review,

Cell motility, adhesion and extracellular matrix degradation / interactions with the microenvironment

TE D

Today, the development of OSCC is seen as a multistep process (Kaur et al., 2013). Besides cell cycle regulation, cancer specific changes in cell motility, adhesion and the extracellular matrix degradation as well as interactions with the stromal microenvironment trigger

EP

carcinogenesis (Luksic et al., 2015; Sen et al., 2016). In tumor progression, cancer-dependent

AC C

changes can modify stromal response, thus promoting invasion and motility of tumor cells (Ahmed Haji Omar et al., 2013). Recently, several genomic copy number changes among OSCC patients could be detected like matrix metalloproteinases (MMPs) genes that play a pivotal role in tumor invasion and metastasis by degrading the extracellular matrix (ECM) (Vincent-Chong et al., 2014) MMPS are zinc-dependent endopeptidases that efficiently degrade the components of the ECM and basement membranes. In this context, almost all members of the MMP family have been implicated in the genesis of various human cancers, including OSCC (Hsin et al., 2014). In this review, four studies could be extracted showing members of the MMP-family as potential biomarker for lymph node metastasis (p=0.006),

ACCEPTED MANUSCRIPT long-term survival (p=0.020), tumor grading (p<0.05) and recurrence (p=0.003) of OSCC (Hsin et al., 2014; Lin et al., 2012; Reis et al., 2011; Vincent-Chong et al., 2014). Interestingly, two studies analyzed MMP in plasma. In general, serum or plasma biomarkers represent a noninvasive, easily accessible method for risk assessment, diagnosis and

RI PT

prognosis (Hsin et al., 2014; Lin et al., 2012). Therefore, MMP may represent a valuable tool in clinical diagnostic pathways of cancer treatment. However, there was no evidence obtainable to prove this.

SC

An intermembranous glycoprotein that is also involved in the communication between the cell

M AN U

and the surrounding matrix is podoplanin. In a study by Inoue et al., it correlated statistically significantly (p=0.020) with tumor grading (Inoue et al., 2012). Concerning the tight junctional complex, the claudin family of integral membrane proteins represents the principal protein assembly. The altered expression of selected members of the claudin family has been reported in several cancers (Sappayatosok et al., 2015; Yoshizawa et al., 2013). Of their 24

TE D

subtypes, claudin 1 and 7 show potential as valid biomarkers for histopathological tumor grading (p< 0.001) and lymph node metastasis (p<0.01, p=0.02 respectively) (Melchers et al.,

EP

2013; Sappayatosok and Phattarataratip 2015; Yoshizawa et al., 2013). E-cadherin, an important cell adhesion molecule and signal transduction factor, can direct the

AC C

formation of protein complexes attached to the actin cytoskeleton. Dynamic changes in cell adhesion due to dislocation of membranous E-cadherin−b-catenin complex lead to loss of epithelial cohesion (Kaur et al., 2013). E-cadherin therefore is an important symbol of occurrence of the loss of epithelial−mesenchymal transition and represents a possible biomarker for tumorigenesis of OSCC (Zhou et al., 2015). Decreased expression is associated with lymph node metastasis (p<0.05) as well as with long-term survival (p=0.007) in OSCC (da Silva et al., 2015; Foschini et al., 2013; Melchers et al., 2013; Zhou et al., 2015). Structural proteins like cytokeratins form the subunits of epithelial intermediary filaments.

ACCEPTED MANUSCRIPT The upregulation of the serum-soluable fragments of these cytokeratines like CYFRA 21-1 can also be seen as a predictor for lymph node metastasis (p=0.012) (Hsu et al., 2015). In summary, there are several biomarkers that show potential in predicting lymph node

RI PT

metastasis and prognosis for patients with OSCC that are associated with cell motility, adhesion and especially extracellular matrix degradation as well as interactions with the

microenvironment. Interestingly, several studies concerning this subject could be reviewed that use liquid biopsy samples from serum samples instead of invasive biopsies. However,

M AN U

SC

here no clinical trial was found to demonstrate their use in clinical routine.

Transcription factors, immunological reactions and angiogenesis In the context of transcription factors, immunological reactions and angiogenesis, some serum markers may also be useful for predicting outcome and tumor recurrence of patients with

TE D

OSCC. Here, squamous cell carcinoma antigen (SCC-Ag) and C-reactive protein (CRP) were validated as strong and independent predictors for long-term survival (p<0.001) as well as tumor recurrence (p<0.001) (Chen et al., 2014; Huang et al., 2012). Furthermore,

EP

angiogenesis plays a crucial role in carcinogenesis (Ziebart et al., 2016). Serum level of PlGF (placenta growth factor), a member of the VEGF (vascular endothelial growth factor) family,

AC C

is associated with long-term survival (p=0.009) (Cheng et al., 2012b). Initiating intracellular signal transduction pathways, VEGF provides the tumor with vessels, oxygen and nutrients and therefore triggers local growth, aggressiveness and metastasis. Several functional singlenucleotide polymorphisms (SNPs) of the VEGF gene have been described and may be used as predictors of long-term survival (p=0.002) (Kammerer et al., 2013). VEGF and its subtypes A and C seem to be valuable, as a solid biopsy and can predict long-term survival (p<0.05) (Yanase et al., 2014). Angiopoietin-like 3 (ANGPTL3), which is involved in new blood vessel growth, is associated with long-term survival shown in in vivo and in vitro analysis

ACCEPTED MANUSCRIPT (p<0.05) (Koyama et al., 2015). Also a statistically significant correlation (p=0.001) between erythropoietin receptor (EPOR), a key factor of angiogenesis, and lymph node metastasis in OSCC could be found.

RI PT

Hypoxia is one of the hallmarks of cancer and shifts the balance of energy productions toward glycolysis (Yang et al., 2015; Ziebart et al., 2011). More than 100 genes involved in pH

regulation, tumor metabolism, angiogenesis, migration and invasion are regulated by hypoxiainducible factor (HIF)−1, a key mediator of the cellular response to hypoxia (Chien et al.,

SC

2012). Among them are isoenzymes of the carbonic anhydrase (CA) family. CA XII was

M AN U

shown to be associated with tumor recurrence (p=0.047), and CA IX with lymph node metastasis (p=0.026). Furthermore, epigenetic modifications cause transcriptional silencing of tumor suppressor genes and therefore trigger malignant transformation (Supic et al., 2011). Here, DNA methylation has been established as an important regulator of carcinogenesis that affects cancer progression, metastatic potential, therapy response and patient survival (Azad

TE D

et al., 2013). Therefore, several transcription factors have been investigated as potential biomarker for OSCC. In our study, we found homeobox protein NKX3-1, WISP-1 (WNT-

EP

inducible-signaling pathway protein 1) and TANGO (transport and Golgi organisation protein 1) among others. It could be demonstrated that these factors significantly initiate lymph node

AC C

metastasis (p<0.001, p=0.05 and p=0.007 respectively) (Clausen et al., 2016; Miyaguchi et al., 2012; Sasahira et al., 2014). miRNAs are a family of small, noncoding RNAs that are involved in the regulation of most cellular processes. Deregulation of miRNAs can therefore trigger carcinogenesis via regulation of the expression levels of oncogenes and tumor suppressor genes (Moratin et al., 2016). Here, mi-RNA9 is closely linked to patient outcome when OSCC has occurred (p=0.009) (Sun et al., 2016). Furthermore, circulating miRNAs are extremely stable in body fluid such as serum, plasma and saliva, which is why they may represent ideal biomarkers. For example, serum levels of miR-483-5p can predict lymph node

ACCEPTED MANUSCRIPT metastasis in patients with OSCC (p<0.01) (Xu et al., 2016). Concerning immunological reactions, epidemiological studies have shown that chronic inflammation increases the risk of cancer (Wang et al., 2014). CD163 is regarded as a highly

RI PT

specific marker for M2 macrophages that could contribute to local immunosuppression and reduced patient survival (p=0.001) as well as lymph node metastasis (p=0.001) (Wang et al., 2014; Weber et al., 2014). In the tumor microenvironment, the balance between pro-

inflammatory activity and anti-tumor immunity triggers proliferation or apoptosis. This

SC

balance is strongly controlled by cytokines such as interleukins (IL) (Kwon et al., 2015). IL-

M AN U

37 was shown to be associated with lymph node metastasis (p=0.006) (Lin et al., 2016), and receptor for IL-4 and IL-6 with tumor recurrence (p=0.002 and p<0.001 respectively) (Kwon et al., 2015; Skrinjar et al., 2015). Furthermore, the B-cell surface molecule BST2 (bone marrow stromal cell antigen 2) is statistically significantly overexpressed in patients with

TE D

OSCC and is associated with long-term survival (p=0.047) (Fang et al., 2014). Taken together, several transcription factors or other molecules that play major roles in immunological reactions and in the process of angiogenesis may function as predictors for

EP

lymph node metastasis, prognosis, tumor recurrence and therapy resistance for patients with OSCC. Especially for tumor recurrence, immune response in the tumor−host interaction

AC C

seems to be fundamental in relapse of OSCC. Besides, this topic seems to be promising as a marker used in liquid biopsy that can minimize invasiveness. However, only prospective studies could be obtained to validate the predictors found in the literature. In contrast to other investigated subjects, numerous studies were found that backed up their in vivo findings with in vitro analyses.

ACCEPTED MANUSCRIPT

CONCLUSIONS In this review, we summarized current evidence of over 100 different biomarkers found for

RI PT

predicting prognosis, outcome and therapy alterations of OSCC. We highlighted the important molecular mechanisms underlying these markers in tissue, blood or saliva samples concerning the following subjects: cell cycle regulation, proliferation and apoptosis; cell motility,

adhesion and extracellular matrix degradation/microenvironment; and transcription factors,

SC

immunologically reactions and angiogenesis. We found several markers that may be used as

M AN U

liquid biopsy samples in predicting tumor aggressiveness, recurrence and resistance to therapy mechanisms. These markers can be obtained noninvasively and therefore represent an interesting tool, especially in early tumor detection as well as in follow-up care of patients with OSCC. In summary, we conclude that there is a lack of clinical trials concerning the predictors evaluated in retrospective and prospective studies. Consequently, we suggest the

AC C

EP

significance.

TE D

initiation of more randomized clinical trials to test these findings regarding their clinical

ACCEPTED MANUSCRIPT Conflict of interest

AC C

EP

TE D

M AN U

SC

RI PT

There are no conflicts of interests.

ACCEPTED MANUSCRIPT REFERENCES Ahmed Haji Omar A, Haglund C, Virolainen S, Hayry V, Atula T, Kontio R, Rihtniemi J, Pihakari A, Salo T, Hagstrom J, Sorsa T: Epithelial and stromal syndecan-1 and -2 are

RI PT

distinctly expressed in oral- and cutaneous squamous cell carcinomas. J Oral Pathol Med 42: 389-395, 2013.

Blatt S, Voelxen N, Sagheb K, Pabst AM, Walenta S, Schroeder T, Mueller-Klieser W,

Ziebart T: Lactate as a predictive marker for tumor recurrence in patients with head and neck

SC

squamous cell carcinoma (HNSCC) post radiation: a prospective study over 15 years. Clin

M AN U

Oral Investig 20: 2097-2104, 2016a.

Blatt S, Ziebart T, Kruger M, Pabst AM: Diagnosing oral squamous cell carcinoma: how much imaging do we really need? A review of the current literature. J Craniomaxillofac Surg, 2016b.

Brazao-Silva MT, Rodrigues MF, Eisenberg AL, Dias FL, de Castro LM, Nunes FD, Faria

TE D

PR, Cardoso SV, Loyola AM, de Sousa SC: Metallothionein gene expression is altered in oral cancer and may predict metastasis and patient outcomes. Histopathology 67: 358-367, 2015.

EP

Brockmeyer P, Jung K, Perske C, Schliephake H, Hemmerlein B: Membrane connexin 43 acts as an independent prognostic marker in oral squamous cell carcinoma. Int J Oncol 45:

AC C

273-281, 2014.

Chen IH, Liao CT, Wang HM, Huang JJ, Kang CJ, Huang SF: Using SCC antigen and CRP levels as prognostic biomarkers in recurrent oral cavity squamous cell carcinoma. PLoS One 9: e103265, 2014.

Cheng AN, Jiang SS, Fan CC, Lo YK, Kuo CY, Chen CH, Liu YL, Lee CC, Chen WS, Huang TS, Wang TY, Lee AY: Increased Cdc7 expression is a marker of oral squamous cell carcinoma and overexpression of Cdc7 contributes to the resistance to DNA-damaging agents. Cancer Lett 337: 218-225, 2013.

ACCEPTED MANUSCRIPT Cheng SJ, Kok SH, Lee JJ, Yen-Ping Kuo M, Cheng SL, Huang YL, Chen HM, Chang HH, Chiang CP: Significant association of SRC protein expression with the progression, recurrence, and prognosis of oral squamous cell carcinoma in Taiwan. Head Neck 34: 13401345, 2012a.

RI PT

Cheng SJ, Lee JJ, Cheng SL, Chen HM, Chang HH, Wang YP, Kok SH, Kuo MY, Chiang CP: Increased serum placenta growth factor level is significantly associated with progression, recurrence and poor prognosis of oral squamous cell carcinoma. Oral Oncol 48: 424-428,

SC

2012b.

Chien MH, Ying TH, Hsieh YH, Lin CH, Shih CH, Wei LH, Yang SF: Tumor-associated

M AN U

carbonic anhydrase XII is linked to the growth of primary oral squamous cell carcinoma and its poor prognosis. Oral Oncol 48: 417-423, 2012.

Chu PY, Hsu NC, Lin SH, Hou MF, Yeh KT: High nuclear protein kinase CbetaII expression is a marker of disease recurrence in oral squamous cell carcinoma. Anticancer Res 32: 3987-

TE D

3991, 2012.

Clausen MJ, Melchers LJ, Mastik MF, Slagter-Menkema L, Groen HJ, van der Laan BF, van Criekinge W, de Meyer T, Denil S, Wisman GB, Roodenburg JL, Schuuring E: Identification

EP

and validation of WISP1 as an epigenetic regulator of metastasis in oral squamous cell carcinoma. Genes Chromosomes Cancer 55: 45-59, 2016.

AC C

da Silva SD, Morand GB, Alobaid FA, Hier MP, Mlynarek AM, Alaoui-Jamali MA, Kowalski LP: Epithelial-mesenchymal transition (EMT) markers have prognostic impact in multiple primary oral squamous cell carcinoma. Clin Exp Metastasis 32: 55-63, 2015. Ding L, Li B, Zhao Y, Fu YF, Hu EL, Hu QG, Ni YH, Hou YY: Serum CCL2 and CCL3 as potential biomarkers for the diagnosis of oral squamous cell carcinoma. Tumour Biol 35: 10539-10546, 2014.

ACCEPTED MANUSCRIPT Fang KH, Kao HK, Chi LM, Liang Y, Liu SC, Hseuh C, Liao CT, Yen TC, Yu JS, Chang KP: Overexpression of BST2 is associated with nodal metastasis and poorer prognosis in oral cavity cancer. Laryngoscope 124: E354-360, 2014. Foschini MP, Leonardi E, Eusebi LH, Farnedi A, Poli T, Tarsitano A, Cocchi R, Marchetti C,

RI PT

Gentile L, Sesenna E, Marucci G, Montebugnoli L: Podoplanin and E-cadherin expression in preoperative incisional biopsies of oral squamous cell carcinoma is related to lymph node metastases. Int J Surg Pathol 21: 133-141, 2013.

SC

Freudlsperger C, Freier K, Hoffmann J, Engel M: Ki-67 expression predicts radiosensitivity in oral squamous cell carcinoma. Int J Oral Maxillofac Surg 41: 965-969, 2012.

M AN U

Fu TY, Hsieh IC, Cheng JT, Tsai MH, Hou YY, Lee JH, Liou HH, Huang SF, Chen HC, Yen LM, Tseng HH, Ger LP: Association of OCT4, SOX2, and NANOG expression with oral squamous cell carcinoma progression. J Oral Pathol Med 45: 89-95, 2016. Gissi DB, Gabusi A, Tarsitano A, Badiali G, Marchetti C, Morandi L, Foschini MP,

TE D

Montebugnoli L: Ki67 Overexpression in mucosa distant from oral carcinoma: a poor prognostic factor in patients with long-term follow-up. J Craniomaxillofac Surg 44: 14301435, 2016.

EP

Goto Y, Kawano S, Matsubara R, Kiyosue T, Hirano M, Jinno T, Maruse Y, Toyoshima T, Kitamura R, Tanaka H, Oobu K, Nakamura S: Possible involvement of DeltaNp63

AC C

downregulation in the invasion and metastasis of oral squamous cell carcinoma via induction of a mesenchymal phenotype. Clin Exp Metastasis 31: 293-306, 2014. Grimm M, Krimmel M, Polligkeit J, Alexander D, Munz A, Kluba S, Keutel C, Hoffmann J, Reinert S, Hoefert S: ABCB5 expression and cancer stem cell hypothesis in oral squamous cell carcinoma. Eur J Cancer 48: 3186-3197, 2012. Hamada T, Nomura M, Kamikawa Y, Yamada N, Batra SK, Yonezawa S, Sugihara K: DF3 epitope expression on MUC1 mucin is associated with tumor aggressiveness, subsequent

ACCEPTED MANUSCRIPT lymph node metastasis, and poor prognosis in patients with oral squamous cell carcinoma. Cancer 118: 5251-5264, 2012a. Hamada T, Wakamatsu T, Miyahara M, Nagata S, Nomura M, Kamikawa Y, Yamada N,

carcinoma. Int J Cancer 130: 1768-1776, 2012b.

RI PT

Batra SK, Yonezawa S, Sugihara K: MUC4: a novel prognostic factor of oral squamous cell

Hanken H, Grobe A, Cachovan G, Smeets R, Simon R, Sauter G, Heiland M, Blessmann M: CCND1 amplification and cyclin D1 immunohistochemical expression in head and neck

SC

squamous cell carcinomas. Clin Oral Investig 18: 269-276, 2014.

Hsin CH, Chen MK, Tang CH, Lin HP, Chou MY, Lin CW, Yang SF: High level of plasma

M AN U

matrix metalloproteinase-11 is associated with clinicopathological characteristics in patients with oral squamous cell carcinoma. PLoS One 9: e113129, 2014.

Hsu YP, Hsieh CH, Chien HT, Lai CH, Tsao CK, Liao CT, Kang CJ, Wang HM, Chang JT, Huang SF: Serum markers of CYFRA 21-1 and C-reactive proteins in oral squamous cell

TE D

carcinoma. World J Surg Oncol 13: 253, 2015.

Huang CF, Zhang L, Ma SR, Zhao ZL, Wang WM, He KF, Zhao YF, Zhang WF, Liu B, Sun

e83479, 2013.

EP

ZJ: Clinical significance of Keap1 and Nrf2 in oral squamous cell carcinoma. PLoS One 8:

Huang SF, Wei FC, Liao CT, Wang HM, Lin CY, Lo S, Huang JJ, Chen IH, Kang CJ, Chien

AC C

HT, Chen HH: Risk stratification in oral cavity squamous cell carcinoma by preoperative CRP and SCC antigen levels. Ann Surg Oncol 19: 3856-3864, 2012. Huber GF, Albinger-Hegyi A, Soltermann A, Roessle M, Graf N, Haerle SK, Holzmann D, Moch H, Hegyi I: Expression patterns of Bmi-1 and p16 significantly correlate with overall, disease-specific, and recurrence-free survival in oropharyngeal squamous cell carcinoma. Cancer 117: 4659-4670, 2011.

ACCEPTED MANUSCRIPT Inoue H, Miyazaki Y, Kikuchi K, Yoshida N, Ide F, Ohmori Y, Tomomura A, Sakashita H, Kusama K: Podoplanin expression during dysplasia-carcinoma sequence in the oral cavity. Tumour Biol 33: 183-194, 2012. Jiang T, Liu G, Wang L, Liu H: Elevated serum Gas6 is a novel prognostic biomarker in

RI PT

patients with oral squamous cell carcinoma. PLoS One 10: e0133940, 2015.

Kamikawa Y, Kanmura Y, Hamada T, Yamada N, Macha MA, Batra SK, Higashi M,

Yonezawa S, Sugihara K: Combination of MUC1 and MUC4 expression predicts clinical

SC

outcome in patients with oral squamous cell carcinoma. Int J Clin Oncol 20: 298-307, 2015. Kammerer PW, Koch FP, Schiegnitz E, Kumar VV, Berres M, Toyoshima T, Al-Nawas B,

M AN U

Brieger J: Associations between single-nucleotide polymorphisms of the VEGF gene and long-term prognosis of oral squamous cell carcinoma. J Oral Pathol Med 42: 374-381, 2013. Kato K, Kawashiri S, Yoshizawa K, Kitahara H, Okamune A, Sugiura S, Noguchi N, Yamamoto E: Expression form of p53 and PCNA at the invasive front in oral squamous cell

TE D

carcinoma: correlation with clinicopathological features and prognosis. J Oral Pathol Med 40: 693-698, 2011.

Kaur J, Sawhney M, DattaGupta S, Shukla NK, Srivastava A, Walfish PG, Ralhan R: Clinical

EP

significance of altered expression of beta-catenin and E-cadherin in oral dysplasia and cancer: potential link with ALCAM expression. PLoS One 8: e67361, 2013.

AC C

Kim JW, Roh JL, Park Y, Cho KJ, Choi SH, Nam SY, Kim SY: Cytoplasmic iASPP expression as a novel prognostic indicator in oral cavity squamous cell carcinoma. Ann Surg Oncol 22: 662-669, 2015.

Klimowicz AC, Bose P, Nakoneshny SC, Dean M, Huang L, Chandarana S, Magliocco AM, Wayne Matthews T, Brockton NT, Dort JC: Basal Ki67 expression measured by digital image analysis is optimal for prognostication in oral squamous cell carcinoma. Eur J Cancer 48: 2166-2174, 2012.

ACCEPTED MANUSCRIPT Kono M, Watanabe M, Abukawa H, Hasegawa O, Satomi T, Chikazu D: Cyclo-oxygenase-2 expression is associated with vascular endothelial growth factor C expression and lymph node metastasis in oral squamous cell carcinoma. J Oral Maxillofac Surg 71: 1694-1702, 2013. Koyama T, Ogawara K, Kasamatsu A, Okamoto A, Kasama H, Minakawa Y, Shimada K,

RI PT

Yokoe H, Shiiba M, Tanzawa H, Uzawa K: ANGPTL3 is a novel biomarker as it activates ERK/MAPK pathway in oral cancer. Cancer Med 4: 759-769, 2015.

Kruger M, Amort J, Wilgenbus P, Helmstadter JP, Grechowa I, Ebert J, Tenzer S, Moergel

SC

M, Witte I, Horke S: The anti-apoptotic PON2 protein is Wnt/beta-catenin-regulated and correlates with radiotherapy resistance in OSCC patients. Oncotarget, 2016.

M AN U

Kruger M, Pabst AM, Al-Nawas B, Horke S, Moergel M: Paraoxonase-2 (PON2) protects oral squamous cell cancer cells against irradiation-induced apoptosis. J Cancer Res Clin Oncol 141: 1757-1766, 2015a.

Kruger M, Pabst AM, Mahmoodi B, Becker B, Kammerer PW, Koch FP: The impact of

TE D

GSTM1/GSTT1 polymorphism for the risk of oral cancer. Clin Oral Investig 19: 1791-1797, 2015b.

Kwon M, Kim JW, Roh JL, Park Y, Cho KJ, Choi SH, Nam SY, Kim SY, Lee BH:

EP

Recurrence and cancer-specific survival according to the expression of IL-4Ralpha and IL13Ralpha1 in patients with oral cavity cancer. Eur J Cancer 51: 177-185, 2015.

AC C

Li Y, Zhang J, Hong S: ANO1 as a marker of oral squamous cell carcinoma and silencing ANO1 suppresses migration of human scc-25 cells. Medicina Oral Patología Oral y Cirugia Bucal: e313-e319, 2014.

Lin CS, Lin YC, Adebayo BO, Wu A, Chen JH, Peng YJ, Cheng MF, Lee WH, Hsiao M, Chao TY, Yeh CT: Silencing JARID1B suppresses oncogenicity, stemness and increases radiation sensitivity in human oral carcinoma. Cancer Lett 368: 36-45, 2015a.

ACCEPTED MANUSCRIPT Lin CW, Tseng SW, Yang SF, Ko CP, Lin CH, Wei LH, Chien MH, Hsieh YS: Role of lipocalin 2 and its complex with matrix metalloproteinase-9 in oral cancer. Oral Dis 18: 734740, 2012. Lin L, Wang J, Liu D, Liu S, Xu H, Ji N, Zhou M, Zeng X, Zhang D, Li J, Chen Q:

RI PT

Interleukin-37 expression and its potential role in oral leukoplakia and oral squamous cell carcinoma. Sci Rep 6: 26757, 2016.

Lin WT, Shieh TM, Yang LC, Wang TY, Chou MY, Yu CC: Elevated Lin28B expression is

SC

correlated with lymph node metastasis in oral squamous cell carcinomas. J Oral Pathol Med 44: 823-830, 2015b.

M AN U

Liu J, Zhang M, Rose B, Veillard AS, Jones D, Zhang X, Soon Lee C, Milross C, Hong A: Ki67 Expression has prognostic significance in relation to human papillomavirus status in oropharyngeal squamous cell carcinoma. Ann Surg Oncol 22: 1893-1900, 2015. Luksic I, Suton P, Manojlovic S, Virag M, Petrovecki M, Macan D: Significance of

TE D

myofibroblast appearance in squamous cell carcinoma of the oral cavity on the occurrence of occult regional metastases, distant metastases, and survival. Int J Oral Maxillofac Surg 44: 1075-1080, 2015.

EP

Ma HL, Jin SF, Tao WJ, Zhang ML, Zhang ZY: Overexpression of stathmin/oncoprotein 18 correlates with poorer prognosis and interacts with p53 in oral squamous cell carcinoma. J

AC C

Craniomaxillofac Surg, 2016. Mascolo M, Ilardi G, Romano MF, Celetti A, Siano M, Romano S, Luise C, Merolla F, Rocco A, Vecchione ML, De Rosa G, Staibano S: Overexpression of chromatin assembly factor-1 p60, poly(ADP-ribose) polymerase 1 and nestin predicts metastasizing behaviour of oral cancer. Histopathology 61: 1089-1105, 2012. Melchers LJ, Bruine de Bruin L, Schnell U, Slagter-Menkema L, Mastik MF, de Bock GH, van Dijk BA, Giepmans BN, van der Laan BF, van der Wal JE, Roodenburg JL, Schuuring E:

ACCEPTED MANUSCRIPT Lack of claudin-7 is a strong predictor of regional recurrence in oral and oropharyngeal squamous cell carcinoma. Oral Oncol 49: 998-1005, 2013. Miyaguchi K, Uzawa N, Mogushi K, Takahashi K, Michikawa C, Nakata Y, Sumino J, Okada N, Mizushima H, Fukuoka Y, Tanaka H: Loss of NKX3-1 as a potential marker for an

carcinoma. Int J Oncol 40: 1907-1914, 2012.

RI PT

increased risk of occult lymph node metastasis and poor prognosis in oral squamous cell

Morandi L, Gissi D, Tarsitano A, Asioli S, Monti V, Del Corso G, Marchetti C,

SC

Montebugnoli L, Foschini MP: DNA methylation analysis by bisulfite next-generation

sequencing for early detection of oral squamous cell carcinoma and high-grade squamous

M AN U

intraepithelial lesion from oral brushing. J Craniomaxillofac Surg 43: 1494-1500, 2015. Moratin J, Hartmann S, Brands R, Brisam M, Mutzbauer G, Scholz C, Seher A, MullerRichter U, Kubler AC, Linz C: Evaluation of miRNA-expression and clinical tumour parameters in oral squamous cell carcinoma (OSCC). J Craniomaxillofac Surg 44: 876-881,

TE D

2016.

Moura IM, Delgado ML, Silva PM, Lopes CA, do Amaral JB, Monteiro LS, Bousbaa H: High CDC20 expression is associated with poor prognosis in oral squamous cell carcinoma. J Oral

EP

Pathol Med 43: 225-231, 2014.

Murali A, Varghese BT, Kumar RR, Kannan S: Combination of genetic variants in cyclin D1

AC C

and retinoblastoma genes predict clinical outcome in oral cancer patients. Tumour Biol 37: 3609-3617, 2016.

Nagata M, Kurita H, Uematsu K, Ogawa S, Takahashi K, Hoshina H, Takagi R: Diagnostic value of cyclin-dependent kinase/cyclin-dependent kinase inhibitor expression ratios as biomarkers of locoregional and hematogenous dissemination risks in oral squamous cell carcinoma. Mol Clin Oncol 3: 1007-1013, 2015. Oliveira-Costa JP, de Carvalho AF, da Silveira da GG, Amaya P, Wu Y, Park KJ, Gigliola MP, Lustberg M, Buim ME, Ferreira EN, Kowalski LP, Chalmers JJ, Soares FA, Carraro

ACCEPTED MANUSCRIPT DM, Ribeiro-Silva A: Gene expression patterns through oral squamous cell carcinoma development: PD-L1 expression in primary tumor and circulating tumor cells. Oncotarget 6: 20902-20920, 2015. Ota N, Ohno J, Seno K, Taniguchi K, Ozeki S: In vitro and in vivo expression of aldehyde

RI PT

dehydrogenase 1 in oral squamous cell carcinoma. Int J Oncol 44: 435-442, 2014.

Reis PP, Waldron L, Perez-Ordonez B, Pintilie M, Galloni NN, Xuan Y, Cervigne NK,

Warner GC, Makitie AA, Simpson C, Goldstein D, Brown D, Gilbert R, Gullane P, Irish J,

SC

Jurisica I, Kamel-Reid S: A gene signature in histologically normal surgical margins is predictive of oral carcinoma recurrence. BMC Cancer 11: 437, 2011.

M AN U

Safi AF, Nickenig HJ, Rothamel D, Zirk M, Thiele O, Grandoch A, Scheer M, Zinser M, Zoller J, Drebber U, Kreppel M: Expression of ezrin in oral squamous cell carcinoma: prognostic impact and clinicopathological correlations. J Craniomaxillofac Surg 43: 18991905, 2015.

TE D

Sagheb K, Blatt S, Rahimi-Nedjat RK, Eigenbrodt S, Al-Nawas B, Walter C: Cervical metastases behavior of T1-2 squamous cell cancer of the oral mucosa. Clin Oral Investig, 2016.

EP

Sappayatosok K, Phattarataratip E: Overexpression of claudin-1 is associated with advanced clinical stage and invasive pathologic characteristics of oral squamous cell carcinoma. Head

AC C

Neck Pathol 9: 173-180, 2015. Sartini D, Pozzi V, Renzi E, Morganti S, Rocchetti R, Rubini C, Santarelli A, Lo Muzio L, Emanuelli M: Analysis of tissue and salivary nicotinamide N-methyltransferase in oral squamous cell carcinoma: basis for the development of a noninvasive diagnostic test for early-stage disease. Biol Chem 393: 505-511, 2012. Sasahira T, Kirita T, Yamamoto K, Ueda N, Kurihara M, Matsushima S, Bhawal UK, Bosserhoff AK, Kuniyasu H: Transport and Golgi organisation protein 1 is a novel tumour progressive factor in oral squamous cell carcinoma. Eur J Cancer 50: 2142-2151, 2014.

ACCEPTED MANUSCRIPT Schiegnitz E, Kammerer PW, Koch FP, Kruger M, Berres M, Al-Nawas B: GDF 15 as an anti-apoptotic, diagnostic and prognostic marker in oral squamous cell carcinoma. Oral Oncol 48: 608-614, 2012. Seki S, Fujiwara M, Matsuura M, Fujita S, Ikeda H, Asahina I, Ikeda T: Prediction of

RI PT

outcome of patients with oral squamous cell carcinoma using vascular invasion and the

strongly positive expression of vascular endothelial growth factors. Oral Oncol 47: 588-593, 2011.

SC

Sen S, Carnelio S: Expression of epithelial cell adhesion molecule (EpCAM) in oral squamous cell carcinoma. Histopathology 68: 897-904, 2016.

M AN U

Shen L, Zhang R, Sun Y, Wang X, Deng AM, Bi L: Overexpression of HSBP1 is associated with resistance to radiotherapy in oral squamous epithelial carcinoma. Med Oncol 31: 990, 2014.

Shi Z, Liu Y, Johnson JJ, Stack MS: Urinary-type plasminogen activator receptor (uPAR)

161, 2011.

TE D

modulates oral cancer cell behavior with alteration in p130cas. Mol Cell Biochem 357: 151-

Shiiba M, Shinozuka K, Saito K, Fushimi K, Kasamatsu A, Ogawara K, Uzawa K, Ito H,

EP

Takiguchi Y, Tanzawa H: MicroRNA-125b regulates proliferation and radioresistance of oral squamous cell carcinoma. Br J Cancer 108: 1817-1821, 2013.

AC C

Shin WJ, Cho YA, Kang KR, Kim JH, Hong SD, Lee JI, Hong SP, Yoon HJ: KiSS-1 expression in oral squamous cell carcinoma and its prognostic significance. APMIS 124: 291298, 2016.

Skrinjar I, Brailo V, Vidovic-Juras D, Vucicevic-Boras V, Milenovic A: Evaluation of pretreatment serum interleukin-6 and tumour necrosis factor alpha as a potential biomarker for recurrence in patients with oral squamous cell carcinoma. Medicina Oral Patología Oral y Cirugia Bucal: e402-e407, 2015.

ACCEPTED MANUSCRIPT Sun L, Liu L, Fu H, Wang Q, Shi Y: Association of decreased expression of serum miR-9 with poor prognosis of oral squamous cell carcinoma patients. Medical Science Monitor 22: 289-294, 2016. Supic G, Kozomara R, Jovic N, Zeljic K, Magic Z: Hypermethylation of RUNX3 but not

RI PT

WIF1 gene and its association with stage and nodal status of tongue cancers. Oral Dis 17: 794-800, 2011.

Tang JY, Hsi E, Huang YC, Hsu NC, Chu PY, Chai CY: High LC3 expression correlates with

SC

poor survival in patients with oral squamous cell carcinoma. Hum Pathol 44: 2558-2562, 2013.

M AN U

Tang JY, Hsi E, Huang YC, Hsu NC, Yang WC, Chang HW, Chai CY, Chu PY: Overexpression of autophagy-related 16-like 1 in patients with oral squamous cell carcinoma. Pathol Oncol Res 21: 301-305, 2015.

Vincent-Chong VK, Salahshourifar I, Karen-Ng LP, Siow MY, Kallarakkal TG, Ramanathan

TE D

A, Yang YH, Khor GH, Rahman ZA, Ismail SM, Prepageran N, Mustafa WM, Abraham MT, Tay KK, Cheong SC, Zain RB: Overexpression of MMP13 is associated with clinical

2014.

EP

outcomes and poor prognosis in oral squamous cell carcinoma. Sci World J 2014: 897523,

Wang S, Sun M, Gu C, Wang X, Chen D, Zhao E, Jiao X, Zheng J: Expression of CD163,

AC C

interleukin-10, and interferon-gamma in oral squamous cell carcinoma: mutual relationships and prognostic implications. Eur J Oral Sci 122: 202-209, 2014. Weber M, Buttner-Herold M, Hyckel P, Moebius P, Distel L, Ries J, Amann K, Neukam FW, Wehrhan F: Small oral squamous cell carcinomas with nodal lymphogenic metastasis show increased infiltration of M2 polarized macrophages─an immunohistochemical analysis. J Craniomaxillofac Surg 42: 1087-1094, 2014.

ACCEPTED MANUSCRIPT Wu HM, Cao W, Ye D, Ren GX, Wu YN, Guo W: Contactin 1 (CNTN1) expression associates with regional lymph node metastasis and is a novel predictor of prognosis in patients with oral squamous cell carcinoma. Mol Med Rep 6: 265-270, 2012. Xia F, Xu JC, Zhang P, Zhang YY, Zhang QW, Chao ZH, Wang F: Glucose-regulated protein

significance. World J Surg Oncol 12: 121, 2014.

RI PT

78 and heparanase expression in oral squamous cell carcinoma: correlations and prognostic

Xie H, Huang S, Li W, Zhao H, Zhang T, Zhang D: Upregulation of Src homology

SC

phosphotyrosyl phosphatase 2 (Shp2) expression in oral cancer and knockdown of Shp2

Oral Radiol 117: 234-242, 2014.

M AN U

expression inhibit tumor cell viability and invasion in vitro. Oral Surg Oral Med Oral Pathol

Xu H, Yang Y, Zhao H, Yang X, Luo Y, Ren Y, Liu W, Li N: Serum miR-483-5p: a novel diagnostic and prognostic biomarker for patients with oral squamous cell carcinoma. Tumour Biol 37: 447-453, 2016.

TE D

Yanase M, Kato K, Yoshizawa K, Noguchi N, Kitahara H, Nakamura H: Prognostic value of vascular endothelial growth factors A and C in oral squamous cell carcinoma. J Oral Pathol Med 43: 514-520, 2014.

EP

Yang CZ, Ma J, Zhu DW, Liu Y, Montgomery B, Wang LZ, Li J, Zhang ZY, Zhang CP, Zhong LP: GDF15 is a potential predictive biomarker for TPF induction chemotherapy and

AC C

promotes tumorigenesis and progression in oral squamous cell carcinoma. Ann Oncol 25: 1215-1222, 2014.

Yang JS, Lin CW, Chuang CY, Su SC, Lin SH, Yang SF: Carbonic anhydrase IX overexpression regulates the migration and progression in oral squamous cell carcinoma. Tumour Biol 36: 9517-9524, 2015. Yoshizawa K, Nozaki S, Kato A, Hirai M, Yanase M, Yoshimoto T, Kimura I, Sugiura S, Okamune A, Kitahara H, Noguchi N, Kato K, Ueki K, Kawashiri S: Loss of claudin-7 is a

ACCEPTED MANUSCRIPT negative prognostic factor for invasion and metastasis in oral squamous cell carcinoma. Oncol Rep 29: 445-450, 2013. Zhang M, Li J, Wang L, Tian Z, Zhang P, Xu Q, Zhang C, Wei F, Chen W: Prognostic significance of p21, p27 and survivin protein expression in patients with oral squamous cell

RI PT

carcinoma. Oncol Lett 6: 381-386, 2013.

Zhong LP, Zhu DW, William WN, Jr., Liu Y, Ma J, Yang CZ, Yang X, Wang LZ, Li J,

Myers JN, Lee JJ, Zhang CP, Zhang ZY: Elevated cyclin D1 expression is predictive for a

SC

benefit from TPF induction chemotherapy in oral squamous cell carcinoma patients with advanced nodal disease. Mol Cancer Ther 12: 1112-1121, 2013.

M AN U

Zhou J, Tao D, Xu Q, Gao Z, Tang D: Expression of E-cadherin and vimentin in oral squamous cell carcinoma. Int J Clin Exp Pathol 8: 3150-3154, 2015. Ziebart T, Blatt S, Gunther C, Volxen N, Pabst A, Sagheb K, Kuhl S, Lambrecht T: Significance of endothelial progenitor cells (EPC) for tumorigenesis of head and neck

TE D

squamous cell carcinoma (HNSCC): possible marker of tumor progression and neovascularization? Clin Oral Investig 20: 2293-2300, 2016. Ziebart T, Walenta S, Kunkel M, Reichert TE, Wagner W, Mueller-Klieser W: Metabolic and

EP

proteomic differentials in head and neck squamous cell carcinomas and normal gingival

AC C

tissue. J Cancer Res Clin Oncol 137: 193-199, 2011.

ACCEPTED MANUSCRIPT

Table 1. Cell cycle regulation, proliferation and apoptosis Study

Sample type

Main parameter

Biomarker

Mechanism

n

p-Value

(Kato, Kawashiri et al. 2011)

In vivo

Tumor biopsy

Tumor recurrence

p53⁄PCNA

Over expression

59

< 0.05

(Cheng, Kok et al. 2012)

In vivo

Tumor biopsy

Long term survival Src protein

Over expression

93

0.00267

(Chu, Hsu et al. 2012)

In vivo

Tumor biopsy

Tumor recurrence

PKCβII

Over expression

59

0.016

(Freudlsperger, Freier et al. In vivo 2012)

Tumor biopsy

Radiation resistance

Ki-67

Under expression 52

0.048

(Hamada, Nomura et al. 2012)

In vivo

Tumor biopsy

LN metastasis

DF3/MUC1

(Hamada, Wakamatsu et al. 2012)

In vivo

Tumor biopsy

(Huang, Cheng et al. 2012) In vivo (Klimowicz, Bose et al. 2012) (Mascolo, Ilardi et al. 2012)

M AN U

SC

RI PT

Reference

206

0.002

Long term survival MUC4

Over expression

150

0.0001

Tumor biopsy

Long term survival Cyclin D1

Over expression

264

0.006

In vivo

Tumor biopsy

Long term survival Ki67

Under expression 121

0.006

In vivo

Tumor biopsy

Long term survival CAF-1⁄ p60 PARP-1 Nestin

Over expression Over expression Over expression

66

< 0.0001

(Michifuri, Hirohashi et al. In vivo 2012)

Tumor biopsy

LN metastasis

ALDH-1 SOX-2

Over expression

80

0.0017 < 0.001

(Rahmani, Alzohairy et al. 2012)

In vivo

Tumor biopsy

LN metastasis

PTEN Bcl2

Under expression 60 Over expression

(Cheng, Jiang et al. 2013)

In vivo

Tumor biopsy

Long term survival Cdc7

Over expression

80

0.01

(Cheng, Liu et al. 2013)

In vivo

Tumor biopsy

Long term survival G 12

Over expression

100

0.009

AC C

EP

TE D

Over expression

< 0.05 < 0.05

(Huang, Zhang et al. 2013) In vivo

Tumor biopsy

Long term survival Keap1 Nrf2

Over expression

(Kaur, Sawhney et al. 2013)

In vivo

Tumor biopsy

Tumor recurrence

E-cadherin b-catenin

Under expression 105 Under expression

(Li, Wang et al. 2013)

In vivo

Tumor biopsy

Tumor recurrence

p-AktThr308

Over expression

(Tang, Hsi et al. 2013)

In vivo

Tumor biopsy

Tumor recurrence

ATG9A

Over expression

90

0.026

(Tang, Hsi et al. 2013)

In vivo

Tumor biopsy

Long term survival LC3

Over expression

90

0.0001

(Zhang, Li et al. 2013)

In vivo

Tumor biopsy

Long term survival p21 p27 survivin

Over expression Over expression Over expression

110

0.0222 0.27 0.58

(Zhong, Zhu et al. 2013)

In vivo

Tumor biopsy

Chemo resistance

Under expression 232

0.001

(Brockmeyer, Jung et al. 2014)

In vivo

Tumor biopsy

Long term survival Connexin 43

Over expression

35

0.0088

(Grimm, Munz et al. 2014) In vivo

Tumor biopsy

161

.0002

In vivo

Tumor biopsy

Long term survival GLUT-1 /TKTL1 Long term survival Cyclin D1

Over expression

(Hanken, Grobe et al. 2014)

TE D

ACCEPTED MANUSCRIPT

Over expression

222

0.0127

(Moura, Delgado et al. 2014)

In vivo

Tumor biopsy

Long term survival CDC20

Over expression

65

0.032

(Ota, Ohno et al. 2014)

In vivo/ In vitro

Tumor biopsy Cell line

Local recurrence

Over expression

90

0.006

(Silva, Alaoui-Jamali et al. In vivo 2014)

Tumor biopsy

Long term survival ErbB1/ErbB4 Over expression

82

0.0013

In vivo

Tumor biopsy

Long term survival NOD1 RIP2

(Wang, Jiang et al. 2014)

AC C

ALDH1

> 0.05

RI PT 191

SC

M AN U

EP

Cyclin D1

43

Under expression 60 Under expression

0.001 < 0.001 0.008

0.006 < 0.001

ACCEPTED MANUSCRIPT

Caspase12 hbD1/2/3 Long term survival GRP78 HPA

Under expression Under expression Over expression 46 Over expression

< 0.001 < 0.001 0.002 0.000

In vivo

Tumor biopsy

(Xie, Huang et al. 2014)

In vivo/ in vitro

Tumor biopsy Cell line

LN metastasis

Shp2

Over expression

88

0.010

(Yang, Ma et al. 2014)

In vivo In vitro

Tumor biopsy Cell line

Chemo resistance

GDF15

Over expression

256

0.019

(Brazao-Silva, Rodrigues et al. 2015)

In vivo

Tumor biopsy

LN metastasis

MT3

Under expression 35

(de Vicente, Santamarta et al. 2015)

In vivo

Tumor biopsy

Long term survival D2-40

Over expression

92

0.08

(Kamikawa, Kanmura et al. 2015)

In vivo

Tumor biopsy

Long term survival MUC-1 /MUC-4

Over expression

206

0.0019

(Kim, Roh et al. 2015)

In vivo

Tumor biopsy

Long term survival iASPP

Over expression

186

< 0.05

(Lee, Kang et al. 2015)

In vivo

Tumor biopsy

Long term survival NANOG p53 CD44

Over expression Over expression Over expression

57

0.019 0.016 0.236

(Liu, Zhang et al. 2015)

In vivo

Tumor biopsy

Long term survival Ki-67

Over expression

105

0.002

(Nagata, Kurita et al. 2015)

In vivo

Tumor biopsy

Local recurrence

CDK2/ CDKN1A CDK1/ CDKN1B

Over expression

77

0.019

Over expression

0.047

0.026

AC C

EP

TE D

M AN U

SC

RI PT

(Xia, Xu et al. 2014)

0.03

(Oliveira-Costa, de Carvalho et al. 2015)

In vivo

Tumor biopsy

LN metastasis

PDL-1

Under expression 142

(Tang, Hsi et al. 2015)

In vivo

Tumor biopsy

LN metastasis

ATG16L1

Over expression

90

0.004

(Fu, Hsieh et al. 2016,

In vivo

Tumor biopsy

Long term survival SOX2

Over expression

436

0.002

ACCEPTED MANUSCRIPT

In vivo

Tumor biopsy

Long term survival Podoplanin/ MMP-9

Over expression

60

0.008

(Murali, Varghese et al. 2016)

In vivo

Tumor biopsy

Long term survival Cyclin D1/ Rb-1 1

Over expression

311

0.029

(Velmurugan, Yeh et al. 2016)

In vivo

Tumor biopsy

Long term survival ANP32A

Over expression

259

0.049

(Yang, Ho et al. 2016)

In vivo

Tumor biopsy

Long term survival Cathepsin B

Over expression

280

0.097

AC C

EP

TE D

M AN U

SC

RI PT

Monteiro, Delgado et al. 2016)

ACCEPTED MANUSCRIPT

Table 2. Cell motility, adhesion and extracellular matrix degradation/microenvironment Main parameter

Biomarker

Mechanism

n

(Reis, Waldron et al. 2011) In vivo

Tumor biopsy

Tumor recurrence

MMP1 COL4A1 P4HA2 THBS2

Over expression

(Yoshizawa, Nozaki et al. 2011)

In vivo

Tumor biopsy

Long term survival uPA+/uPAR+ Over expression 54 Under expression /maspin-

(Fujii, Shomori et al. 2012) In vivo

Tumor biopsy

Long term survival grade 2 CAF CD163+ macrophage

Over expression

p-Value

RI PT

Sample type

199

SC

Study

M AN U

Reference

108

0.003

0.02

0.003 0.007

Over expression

In vivo In vitro

Tumor biopsy and cell line

LN metastasis

ABCB5

Over expression

191

0.0002

(Inoue, Miyazaki et al. 2012)

In vivo

Tumor biopsy

Grading

podoplanin

Over expression

69

0.020

(Lin, Tseng et al. 2012)

In vivo

Blood sample

Advanced clinical stage

LCN2/MMP- Over expression 9

195

<0.05

(Wushou, Pan et al. 2012)

In vivo

Tumor biopsy

LN metastasis

Twist

Over expression

60

0.012

(Ahmed Haji Omar, Haglund et al. 2013)

In vivo

Tumor biopsy

Long term survival Syndecan-1

Over expression

35

0.015

(Foschini, Leonardi et al. 2013)

In vivo

Tumor biopsy

LN metastasis

E-cadherin Podolopin

Under expression 102 Under expression

(Kono, Watanabe et al. 2013)

In vivo

Tumor biopsy

LN metastasis

VEGF-C

Over expression

(Melchers, Bruine de Bruin et al. 2013)

In vivo

Tumor biopsy

Differentiation grade

Claudin 7

Under expression 227

AC C

EP

TE D

(Grimm, Krimmel et al. 2012)

60

<0.05

< .01

< 0.001

ACCEPTED MANUSCRIPT

In vivo

Tumor biopsy

LN metastasis

ITGA3 ITGB4

Over expression Over expression

(Yoshizawa, Nozaki et al. 2013)

In vivo In vitro

Tumor biopsy Cell line

LN metastasis

Claudin-7

Under expression 67

(Dhanda, Triantafyllou et al. 2014)

In vivo

Tumor biopsy

Long term survival SMA /SERPINE1

Over expression

102

<0.001

(Ding, Li et al. 2014)

In vivo

Blood sample

Grading

CCL 2

Over expression

98

0.018

(Goto, Kawano et al. 2014) In vivo In vitro

Tumor biopsy Cell line

LN metastasis

DNp63

SC

0.000 0.022

(Nagata, Noman et al. 2013)

Under expression 78

<0.01

(Hsin, Chen et al. 2014)

In vivo

Blood sample

LN metastasis

MMP-11

Over expression

330

0.006

(Li, Zhang et al. 2014)

In vivo In vitro

Tumor biopsy Cell line

LN metastasis

ANO1

Over expression

160

<0.05

(Magnussen, Rikardsen et al. 2014)

In vivo

Tumor biopsy

LN metastasis

uPAR PAI-1

Under expression 115

0.031 0.021

(Natarajan, Hunter et al. 2014)

In vivo

Tumor biopsy

LN metastasis

S100A4

Over expression

47

<0.001

(Vincent-Chong, Salahshourifar et al. 2014)

In vivo

Tumor biopsy

Long term survival MMP13

Over expression

68

0.020

(Bose, Brockton et al. 2015)

In vivo

Tumor biopsy

Long term survival nFD

Over expression

107

0.002

(Byatnal, Byatnal et al. 2015)

In vivo

Tumor biopsy

LN metastasis

Over expression

75

0.032

(da Silva, Morand et al. 2015)

In vivo

Tumor biopsy

Long term survival E-cadherin b-catenin

(Hsu, Hsieh et al. 2015)

In vivo

Blood sample

LN metastasis

<0.01

RI PT

M AN U

TE D

EP

AC C

COX-2

270

Under expression 102

0.007

130

0.012

CYFRA 21-1 Over expression

ACCEPTED MANUSCRIPT

Blood sample

Long term survival Gas6

Over expression

128

< 0.001

(Kong, Syed Zanaruddin et In vivo al. 2015)

Tumor biopsy

Long term survival TWIST1/ ZEB2

Over expression

87

0.025

(Luksic, Suton et al. 2015)

In vivo

Tumor biopsy

Long term survival myofibroblast Over expression

(Ni, Ding et al. 2015)

In vivo

Tumor biopsy

LN metastasis

CD68+ TAMs

Over expression

(Sappayatosok and Phattarataratip 2015)

In vivo

Tumor biopsy

Ln metastasis

Claudin-1

Over expression

45

0.02

(Zhang, Zhang et al. 2015) In vivo In vitro

Tumor biopsy Cell line

LN metastasis

CMTM3

Over expression

201

<0.05

(Zhou, Tao et al. 2015)

Tumor biopsy

LN metastasis

E-cadherin

Under expression 42

152

0.009

91

0.034

SC

M AN U

TE D EP AC C

In vivo

RI PT

In vivo

(Jiang, Liu et al. 2015)

0.000

ACCEPTED MANUSCRIPT

Table 3. Transcription factors, immulogical reactions and angiogenesis Study

Sample type

Main parameter

(Huber, Albinger-Hegyi et al. 2011)

In vivo

Tumor biopsy

Long term survival Bmi-1/ p16

Under expression

252

0.002

(Lin, Chen et al. 2011)

In vivo

Tumor biopsy

Long term survival IGF2BP3

Over expression

93

0.0017

(Supic, Kozomara et al. 2011)

In vivo

Tumor biopsy

Long term survival DAPK

Hypermethylation

47

0.007

(Supic, Kozomara et al. 2011)

In vivo

Tumor biopsy

LN metastasis

Hypermethylation

76

0.058

(Cheng, Lee et al. 2012)

In vivo

Blood sample

Long term survival PlGF

Over expression

72

0.009

(Chien, Ying et al. 2012)

In vivo

Tumor biopsy

Tumor recurrence

Over expression

264

0.047

(Huang, Cheng et al. 2012) In vivo

Tumor biopsy

Long term survival EGFR

Over expression

160

0.005

(Huang, Wei et al. 2012)

In vivo

Blood sample

Long term survival SCC-AG/ CRP

Over expression

142

<0.001

(Lin, Chuang et al. 2012)

In vivo

Tumor biopsy

LN metastasis

Over expression

256

0.001

(Miyaguchi, Uzawa et al. 2012)

In vivo

Tumor biopsy

LN metastasis

Under expression

60

<0.001

(Piao, Liu et al. 2012)

In vivo

Tumor biopsy

Long term survival USP 22

Over expression

319

<0.001

(Sartini, Pozzi et al. 2012)

In vivo

Tumor biopsy Saliva sample

LN metastasis

NNMT

Over expression

27 16

<0.05

(Wu, Cao et al. 2012)

In vivo In vitro

Tumor biopsy Cell line

LN metastasis

CNTN1

Over expression

45

0.006

(Chen, Yeh et al. 2013)

In vivo

Tumor biopsy

Long term survival ARK2

Over expression

215

0.005

TE D

CA XII

EPOR

EP

AC C

n

M AN U

RUNX3

Mechanism

SC

Biomarker

NKX3-1

p-Value

RI PT

Ref.

ACCEPTED MANUSCRIPT

In vivo

Blood sample

Long term survival VEGF +405 G/G

Over expression

113

0.002

(Monteiro, Delgado et al. 2013)

In vivo

Tumor biopsy

Long term survival p-mTOR

Over expression

46

0.043

(Seto, Uchida et al. 2013)

In vivo

Tumor biopsy

Long term survival GnT-V

Under expression

68

0.025

(Shiiba, Shinozuka et al. 2013)

In vivo In vitro

Tumor biopsy Cell line

Radioresistance

miR-125b

Over expression

50

<0.05

(Chen, Liao et al. 2014)

In vivo

Blood sample

Tumor recurrence

SCC-AG/ CRP

Over expression

(Fang, Kao et al. 2014)

In vivo In vitro

Tumor biopsy Cell line

Long term survival BST2

(Grimm, Munz et al. 2014) In vivo

Tumor biopsy

Long term survival HP

(Ishige, Kasamatsu et al. 2014)

In vivo

Tumor biopsy

LN metastasis

(Lin, Ho et al. 2014)

In vivo

Tumor biopsy

Tumor recurrence

(Sasahira, Kirita et al. 2014)

In vivo In vitro

Tumor biopsy Cell line

LN metastasis

(Shen, Zhang et al. 2014)

In vivo In vitro

Tumor biopsy Cell line

Radioresistance

(Tanaka, Sho et al. 2014)

In vivo

Tumor biopsy

(Wang, Sun et al. 2014)

In vivo

(Weber, Buttner-Herold et al. 2014) (Xia, Du et al. 2014)

SC

RI PT

(Kammerer, Koch et al. 2013)

<0.001

Over expression

159

0.047

Over expression

191

0.026

Under expression

102

<0.05

Tn/NF-κB

Over expression

143

<0.001

TANGO

Over expression

171

0.0044

Over expression

96

<0.001

Long term survival ETBR

Over expression

107

0.003

Tumor biopsy

Long term survival CD163

Over expression

298

0.001

In vivo

Tumor biopsy

LN metastasis

CD163

Over expression

34

0.001

In vivo

Tumor biopsy

LN metastasis

AEG-1

Over expression

87

<0.001

M AN U

534

EP

TE D

KLK13

AC C

HSBP1

ACCEPTED MANUSCRIPT

In vivo In vitro

Tumor biopsy Cell line

Long term survival VEGF-C

Over expression

61

<0.05

(You, Lin et al. 2014)

In vivo

Tumor biopsy

Tumor recurrence

DEC-1

Over expression

56

<0.05

(Zhu, Tan et al. 2014)

In vivo

Tumor biopsy

Long term survival PLCG1

Over expression

256

0.022

(Koyama, Ogawara et al. 2015)

In vivo In vitro

Tumor biopsy Cell line

Long term survival ANGPTL3

Over expression

109

<0.05

(Kwon, Kim et al. 2015)

In vivo

Tumor biopsy

Tumor recurrence

(Lin, Lin et al. 2015)

In vivo In vitro

Tumor biopsy Cell line

Long term survival JARID1B

(Lin, Shieh et al. 2015)

In vivo In vitro

Tumor biopsy Cell line

LN metastasis

(Skrinjar, Brailo et al. 2015)

In vivo

Blood sample

Tumor recurrence

(Yang, Lin et al. 2015)

In vivo

Tumor biopsy

LN metastasis

(Clausen, Melchers et al. 2016)

In vivo

Tumor biopsy

LN metastasis

(Harada, Izumi et al. 2016) In vivo

Tumor biopsy

Long term survival GalNAc-T3+

(Kim, Park et al. 2016)

In vivo

Tumor biopsy

LN metastasis

(Lin, Wang et al. 2016)

In vivo

Tumor biopsy

LN metastasis

(Sen and Carnelio 2016)

In vivo

Tumor biopsy

Long term survival EpCAM

(Shin, Cho et al. 2016)

In vivo In vitro

Tumor biopsy Cell line

LN metastasis

SC

Overexpression

186

0.002

Over expression

81

0.002

Lin28B

Over expression

60

<0.05

Interleukin-6

Over expression

28

<0.001

CAIX

Over expression

271

0.026

WISP1

Over expression

204

0.05

Over expression

110

<0.001

GCS/ P-gp

Over expression

186

<0.05

IL-37

Under expression

60

0.006

Over expression

60

0.004

Under expression

99

0.007

TE D

M AN U

IL-4Ra

EP

AC C

RI PT

(Yanase, Kato et al. 2014)

KiSS-1

ACCEPTED MANUSCRIPT

(Sun, Liu et al. 2016)

In vivo

Blood sample

Long term survival miR-9

Under expression

104

0.022

(Xu, Yang et al. 2016)

In vivo

Blood sample

LN metastasis

Under expression

101

<0.01

AC C

EP

TE D

M AN U

SC

RI PT

miR-483-5p