Prognostic significance of immunohistochemical biomarkers in oral squamous cell carcinoma

Abstract

Advances in understanding of the molecular mechanisms underlying oral squamous cell carcinoma (OSCC) have resulted in an increasing number of biomarkers that can be used to predict the behaviour of this disease. The authors conducted a literature review of studies examining the role of immunohistochemistry-based protein biomarkers in predicting OSCC outcome. Only articles published in PubMed-indexed journals over the past 5 years were considered. 22 molecular biomarkers were identified and classified into five groups based on their biological functions: cell cycle acceleration and proliferation; tumour suppression and apoptosis; hypoxia; angiogenesis; and cell adhesion and matrix degradation. The cell cycle acceleration and proliferation biomarkers showed the most divergent prognostic findings. Studies on tumour suppression and apoptosis biomarkers were the most prevalent. There were only a few studies examining molecular biomarkers of hypoxia and angiogenesis, and studies examining cell adhesion and matrix degradation biomarkers have shown that this group has the greatest potential for assessing prognostic parameters. Amongst the several proteins analysed, the immunohistochemical expression levels of epithelial growth factor receptor (EGFR), p53, and matrix metalloproteinases (MMPs) have demonstrated the greatest potential for survival prediction in OSCC, but this review demonstrates that their prognostic relevance is debatable and requires further standardisation.

Oral squamous cell carcinoma (OSCC) results from the multistep accumulation of heterogeneous genetic changes in squamous cells. These changes progressively increase the ability of transformed cells to proliferate and invade . The heterogeneity of these changes explains why tumours at the same clinical stage and localisation often show significant differences in their clinical outcomes and treatment responses .

OSCC remains one of the most difficult malignancies to control because of its high propensity for local invasion and cervical lymph node dissemination . The biological factors that underlie the locoregional and distant spreading of these neoplasms are not completely understood . The behaviour of OSCC is difficult to predict solely using conventional clinical and histopathological parameters, and due to location of the disease, the multimodal tumour therapy usually prescribed leads to a reduction in quality of life, making the psychosocial consequences of OSCC greater than other malignancies . For these reasons, despite advances in therapeutic strategies, the survival rate of OSCC patients is still poor.

Investigation of phenotypic changes in OSCC cells may have a strategic prognostic value, and special attention has recently been focussed on the use of potential molecular biomarkers as reliable predictors of tumour aggressiveness . The assessment of prognostic biomarkers can also be useful in the selection of patients who would best benefit from intensive adjuvant therapy .

In recent years, the number of molecular-based assays has increased but histopathology remains the gold standard for most diagnostic and therapeutic decisions. Immunohistochemistry (IHC) is a globally available tool that complements histopathological analysis by detecting gene expression at the protein level. Advances in the understanding of cancer at the regulatory protein expression level have resulted in the identification of some prognostic tumour biomarkers associated with the clinical outcome of OSCC, and there are diverse types of cell and tissue molecular biomarkers that can provide information complementary to that which can be obtained from clinical examination and histopathological studies.

Tumour suppressor genes, oncogenes, cell proliferation markers, angiogenic markers, and cell adhesion molecules have been studied as potential tools to predict the prognosis of patients with OSCC .

An immunohistochemical panel using multiple prognostic molecular biomarkers can provide useful information for the identification of high-risk OSCC patients. The objective of this study is to identify, through an extensive review of recently published literature, the major IHC biomarkers that have been significantly associated with the prognosis of OSCC.

Material and methods

This review investigated the scientific literature examining the role of the major biomarkers associated with the clinical outcome of OSCC over the past 4 years (June 2005 to June 2009). A web-based search was performed in the PubMed database to retrieve studies on this subject. The search terms were ‘oral cancer’, ‘OSCC’, ‘prognostic biomarker’, ‘molecular biomarker’, ‘prognostic factor’, and ‘survival, prediction, outcome, mortality’. This search was subsequently refined, and the research manuscripts were then selected based on the abstract text.

The studies selected for this review were retrospective investigations focussing on prognostic and survival parameters in which IHC was performed in the primary OSCC tumour. In the selected manuscripts, all surgical specimens had been taken prior to the initiation of radiotherapy or chemotherapy. The following anatomical localisations were included in this review: C00, C02–C06, C06.1, and C06.2 . Reports detailing with in vitro investigations were excluded from this review.

Results

The search terms used for this review identified 22 different molecular biomarkers that had been investigated using IHC to assess prognostic parameters in OSCC patients. Table 1 demonstrates the prognostic relevance of all tumour biomarkers divided by group, as well as the variations in sample sizes and in P statistical values according to each specific marker studied amongst the different publications. The P values were obtained from multivariate Cox regression or Kaplan–Meier analysis (preferably overall survival) when the first was lacking. The biomarkers were classified into five groups based on their biological function: cell cycle acceleration and proliferation; tumour suppression and apoptosis; hypoxia; angiogenesis; and cell adhesion and matrix degradation.

Table 1
Prognostic relevance of 22 different biomarkers investigated through IHC in OSCC.
Molecular biomarker Prognostic significance Sample size and P value variation (min.-max.) a No prognostic significance Sample size and P value variation (min.-max.) a Total
Cell cycle acceleration and proliferation
EGFR 5 b 41–140 – 0.0002–0.02 2 47–165 – 0.114 7
Cyclin D1 2 b , c 140–348 – <0.0001–0.0181 2
Ki-67 3 41–113 – 0.0001–<0.001 1 74 – 0.091 4
PCNA 3 60 – 113 – >0.05 3
Akt1 1 84 – 0.013 1
Tumour suppression and apoptosis
p53 and p63 9 b 45–140 – 0.0002–0.049 9
p21 and p27 2 106–192 – 0.018–0.03 1 192 – 0.43 3
Bcl-2 family members 4 35–110 – <0.005–0.0489 1 110 – 0.4901–0.8287 5
pRb 2 220–348 – 0.004–<0.05 2
Survivin 1 78 – 0.002 1
Hypoxia
HIF-1α 3 57–112 – 0.004–0.048 3
CA IX 2 60–117 – 0.02–<0.05 1 68 – 0.062 3
GLUT-1 1 40 – 0.035 1
EPOR 1 43 – 0.030 1
Angiogenesis
VEGF 4 59–1002 – <0.001–0.045 1 84 – 0.168 5
CD105 3 26–176 – <0.001–0.02 3
Ephs 1 59 – 0.037 1
Cell adhesion and matrix degradation
MMPs 6 68–138 – 0.008–0.032 1 38 – data not shown 7
CD44 3 36–138 – 0.01–0.03 3
Cadherins 3 47–67 – <0.0001–<0.05 1 84 – data not shown 4
Catenins 1 135 – 0.0088 1
Versican 1 139 – 0.048 1
Total 58 12 70

a Variations regarding the number of investigated patients and concerning the P statistical value (Cox regression or Kaplan–Meier analysis) according to each specific marker studied amongst the different publications.

b Combined expression of EGFR, p53, and cyclin D1 was associated with an unfavourable prognosis in OSCC patients .

c Tumours with downregulation of p16 and overexpression of cyclin D1 exhibited the worst prognosis .

Cell cycle acceleration and proliferation molecules

Five biomarkers were identified belonging to this group: epithelial growth factor receptor (EGFR); cyclin D1; Ki-67; proliferating cell nuclear antigen (PCNA); and serine/threonine kinase 1 (Akt1) ( Table 1 ).

EGFR (EGF-R, c-erb1–4, Her-2/neu)

EGFR is a transmembrane cell surface receptor that binds to some ligands such as EGF and TGF-α, thereby activating the protein–tyrosine kinase system, which regulates the signalling involved in cell proliferation and differentiation . It belongs to a family of four similar receptors: HER-1 (ErbB1), HER-2 (neu/ErbB2), HER-3 (ErbB3), and HER-4 (ErbB4). EGFR activation can enhance the malignant potential of epithelial tissues . In some studies, EGFR overexpression had been correlated with poor prognostic in OSCC patients , but these results were not confirmed in other investigations . A single study had identified a significant association between the combined expression of EGFR, p53, and cyclin D1 and an unfavourable overall survival (OS) in OSCC patients .

Cyclin D1

Tumour cell proliferation is constantly associated with genetic or epigenetic modifications in key cell cycle molecules. The D-type cyclins are expressed during the progression from G0/G1 to S phase of the mammalian cell cycle . Cyclin D1 is an oncogene that drives cell cycle progression, and the decision for cell growth or arrest may depend on the concentration of cyclin D1 . Cyclin D1 amplification is one of the most frequent molecular alterations in head and neck squamous cell carcinomas (HNSCC) . Cyclin D1 expression alone has not been associated with OSCC progression. S hiraki et al. showed that the combined expression of cyclin D1, EGFR, and p53 was significantly associated with an unfavourable OS in OSCC patients. Similarly, J ayasurya et al. demonstrated the decreased expression of p16, coupled with the overexpression of cyclin D1, in tumours is associated with an unfavourable clinical outcome.

Ki-67

The expression of the human Ki-67 protein is strictly connected with cell proliferation. The fact that the Ki-67 protein is not expressed in G0 resting cells, but is expressed during all active phases of the cell cycle (G1, S, G2, and mitosis), makes it an excellent biomarker for determining the fraction of actively proliferating cells in a given tumour . In an analysis of 113 OSCC patients, M young et al. correlated the expression of Ki-67 expression with the cumulative survival rate, confirming that this biomarker provides useful information in predicting a worse prognosis for OSCC patients. This finding has been corroborated by other authors , whilst L ee et al. have recently observed no independent association between Ki-67 expression and OSCC survival.

PCNA

PCNA is a well-known cell cycle marker protein that plays an important role in nucleic acid metabolism as a component of the replication and repair machinery . In several recent investigations, PCNA expression has not been significantly associated with survival in OSCC patients .

Akt1

Akt plays a pivotal role in cell survival and proliferation through a number of downstream effectors . Akt overexpression was an independent and significant indicator of poor prognosis in a study examining OSCC outcome .

Tumour suppression and apoptosis biomarkers

Five subsets of biomarkers were identified in this group ( Table 1 ): p53/p63, p21/p27, Bcl-2 family members, pRb, and Survivin.

p53 and p63

The p53 gene is one of the most studied biomarkers in OSCC. Functional inactivation of p53 causes defects in DNA repair and apoptosis, with a subsequent increase in genetic instability that can lead to the accumulation of mutations . The high expression of p53 has been associated with a poor prognosis , and the combined expression of p53, cyclin D1, and EGFR has been correlated with an unfavourable OS in OSCC patients . The data examining the prognostic value of the expression of p63, a p53 homologue, is controversial. Some authors have found that overexpression of p63 is associated with better prognosis in OSCC , whilst others have found the opposite .

p21 and p27

Cell cycle progression is regulated by the cyclins and cyclin-dependent kinases (CDKs). The activity of these enzymes is coordinated by the inhibitory action of the Cip/Kip family, including both p21 waf1/cip1 and p27 Kip1 . P21 waf1/cip1 plays an important function in the regulation of the G1-to-S transition of the cell cycle, because it is an inhibitor of the CDKs . The expression of p27 Kip1 can result in cell cycle arrest or progression, thereby regulating cell proliferation, cell motility, and apoptosis. Based on its post-translational modifications, p27 Kip1 can both positively and negatively regulate these processes. The loss of the expression or function of these two G1-checkpoint CDK inhibitors has been implicated in the progression of many human tumours . There are controversial findings concerning the clinical outcome of p21 waf1/cip1 -positivite OSCCs , and no significant association between p27 Kip1 expression and OSCC prognostic has been identified .

Bcl-2 family members

Bcl-2 family members are apoptosis regulatory proteins. This family includes both anti-apoptotic (e.g. Bcl-2, Bcl-X) and pro-apoptotic proteins (e.g. Bax and Bak), and it is the balance between them that determines the cell fate . C amisasca et al. found that the expression of Bcl-2, Bax, and Bcl-X could be correlated with a favourable outcome in OSCC. d e Vicente et al. demonstrated that Bcl-2 protein expression was associated with poor prognosis, and they failed to identify any association between Bax expression and disease outcome. Some investigations have shown that the survival rate was significantly higher in OSCC patients with Bcl-2-negative and Bax-positive tumours .

pRb

The Rb pathway plays a crucial regulatory role in cell cycle progression, and its function can be inhibited by specific mutations. The phosphorylation of pRb occurs following the activation of CDK4 or CDK6 through cyclin D1 and results in its functional inhibition and liberation of transcription factors required for cell cycle progression . Although J ayasurya et al. have demonstrated a significant association between pRb overexpression and reduced disease-free survival (DFS), S oni et al. have shown that OSCCs that lost pRb expression were aggressive carcinomas and had a poor prognosis.

Survivin

Survivin is an inhibitor of apoptosis that generally is undetectable in normal mucosa, but is overexpressed in most head and neck cancers . L o Muzio et al. identified Survivin expression as a potential biomarker of aggressive and invasive OSCCs.

Hypoxia biomarkers

Four IHC hypoxia biomarkers were identified as putative prognostic parameters: hypoxia inducible factor 1α (HIF-1α); carbonic anhydrase IX (CA IX); glucose transporter 1 (GLUT-1); and erythropoietin receptor (EPOR).

HIF-1α

HIF-1 is a heterodimeric transcription factor composed of alpha and beta subunits. The HIF-1α subunit mediates HIF-1 function in response to cellular hypoxia. Under normal oxygen conditions, the HIF-1α subunit is rapidly degraded by the proteasome and has a very short half-life. Under hypoxic conditions, the proteolytic degradation is suppressed, resulting in overexpression of this subunit. In response to decreased oxygen, HIF-1α induces the transactivation of more than 70 genes involved in hypoxia adaptation and/or reversion . The diffuse overexpression of HIF-1α has been associated with a good prognosis in OSCC patients , but some recent investigations have found the opposite result .

Carbonic anhydrase IX

Carbonic anhydrases (CAs) form a large family of genes that encode zinc metalloenzymes. They are involved in the reversible hydration of carbon dioxide to carbonic acid, thereby maintaining a stable intracellular pH. The CAs also participate in other biological processes such as cellular respiration and dystrophic calcification . CA IX is a HIF-1-dependent member of the CA family and a transmembrane glycoprotein involved in pH homeostasis . C hoi et al. have shown that CA IX overexpression is significantly associated with disease recurrence and a worse OS in OSCC patients. K im et al. have demonstrated a statistically significant association between high CA IX expression and a poorer OS in a series of tongue squamous cell carcinomas. In contrast, S akata et al. did not find any association between CA IX expression and DFS.

GLUT-1

The glucose transporters (GLUT) are also regulated via the HIF-1 pathway and can mediate cellular glucose uptake, thereby perpetuating anaerobic glycolysis . It has been suggested that, like CA IX and HIF-1α, GLUT-1 might represent an endogenous marker of hypoxia . GLUT has been identified in diverse human tumours, and there is some evidence that GLUT-1 is related to aggressiveness in HNSCC . The increased expression of GLUT-1 was significantly associated with a shorter OS and radiotherapeutic failure in OSCC .

EPOR

Erythropoietin (EPO) is a glycoprotein hormone that mediates the production of red blood cells. It is synthesised in the kidney in response to hypoxia, and its biological effects are regulated through its interaction with a specific transmembrane EPOR and signalling mediated through HIF-1α . Head and neck cancer cells are known to express EPOR , and R oh et al. have found that high EPOR expression can be associated with a significantly worse prognosis in patients with oral tongue squamous cell carcinoma.

Angiogenesis biomarkers

Three angiogenic biomarkers were identified as possible prognostic parameters ( Table 1 ): vascular endothelial growth factor (VEGF); endoglin (CD105); and Eph receptor tyrosine kinases (Ephs).

VEGF

VEGF acts functions mainly as an angiogenic cytokine that promotes proliferation, differentiation, and migration of vascular endothelial cells. It also induces vessel permeability and promotes endothelial cell survival by preventing apoptosis . One study failed to identify an independent and significant association between VEGF expression and prognostic parameters in patients with OSCC , but VEGF overexpression was identified as an adverse DFS prognosticator in another investigation . Two studies confirmed that patients with VEGF-positive tumours have significantly poorer survival . In a meta-analysis carried out on HNSCC patients (71% of them diagnosed as OSCC), K yzas et al. demonstrated that VEGF overexpression was significantly associated with a worse OS.

CD105

Endoglin is a regulatory component of the cellular transforming growth factor-β (TGF-β) receptor complex and can modulate angiogenesis by the regulation of different cellular functions including proliferation, differentiation, and migration. The assessment of neo-vascularisation using CD105 expression has been considered a potential predictor of prognosis in different solid malignancies . It has been shown that the evaluation of micro-vessel density through CD105 expression in primary OSCC may identify patients at risk of recurrence of the disease or poor outcome following treatment . In another study performed only on biopsies of early tongue squamous cell carcinomas staged as T1 or T2, C huang et al. also demonstrated that CD105 expression was a significant and independent prognostic predictor of a worse prognosis.

Ephs receptors

Ephs receptors (erythropoietin-producing human hepatocellular carcinoma) comprise the largest family of vertebrate receptor tyrosine kinases. Together with their membrane-anchored ephrin ligands (eph family receptor interacting proteins), they form a vital cell–cell communication system capable of bi-directional signalling . This Eph receptor/ephrin system has been shown to play a crucial role in embryonic development, as well as in several human cancers . Investigations suggest that this system can stimulate invasive behaviour in a tumour, thereby promoting a more aggressive and metastatic phenotype . Biochemical and genetic evidence has also implicated Ephs receptors as important regulators of angiogenesis . In a recent study, S hao et al. showed that a high expression of EphA2 was associated with a shorter survival period in tongue squamous cell carcinoma patients.

Cell adhesion and matrix degradation molecules

Five IHC matrix degradation and cell adhesion related molecules were identified as putative prognostic biomarkers associated with OSCC ( Table 1 ): matrix metalloproteinases (MMPs), CD44, cadherins, catenins, and versican.

MMPs

The MMPs are a family of proteases normally expressed by invasive tumours and the adjacent stroma . They are zinc-associated endopeptidases capable of degrading all components of the extracellular matrix (ECM), as well as the basement membrane. MMPs have an essential role in ECM degradation in several situations, including development, inflammation, tissue repair, and tumour invasion and metastasis . There are over 20 known mammalian MMPs , and the expression of MMP family members in OSCC tissues has been widely reported. Although a retrospective study with tongue carcinomas found no correlation between MMP-2 and MMP-9 overexpression and survival , there are several conflicting studies examining the prognostic role of MMPs in OSCC . MMP-9 expression was associated with poor prognosis in a subgroup of early stage OSCC patients without neck lymph node metastases . In cases of OSCC presenting with lymph node metastasis, d e Vicente et al. identified a significant correlation between MMP-7 and MMP-14 (MT1-MMP) expression and poor survival. In another study performed with HNSCC patients (85% of them diagnosed with OSCC), L uukkaa et al. found that a high MMP-13 (collagenase-3) expression level was significantly associated with a short survival time.

CD44

The CD44 family is a widely expressed transmembrane glycoprotein family that binds hyaluronic acid, growth factors, and ECM proteins . The CD44 family consists of a standard form of CD44 (CD44s) and an alternative splice variant (CD44v). CD44 has been shown to be a major factor in cell–cell and cell–ECM interactions, including cell migration, adhesion, and some lymphocyte functions . The low expression of CD44s on tumour cells has been significantly correlated with poor prognosis in tongue carcinomas , and the absence of CD44v expression has been associated with a shorter survival time in lip OSSC . Similarly, K osunen et al. has shown that the irregular expression of CD44 in OSCC was correlated with poor DFS and OS.

Cadherins

Cadherins are a family of transmembrane glycoproteins involved in cell–cell adhesion . In most epithelial cells, this adhesion is established and maintained by the epithelial-cadherin (E-cad) complex, localised mainly in the zonula adherens junctions. The cadherin family also includes more than 20 proteins, such as placental cadherin (P-cad), that have been described in several tissues and organs . E-cad is expressed in the intercellular junctions of keratinocytes, whilst P-cad is predominantly detected on the surface of basal keratinocytes in the normal epidermis. Therefore, cells migrating into the suprabasal compartment tend to down-regulate P-cad expression . The loss of adhesion resulting from the reduced expression of cadherins can play an important role in tumour invasion and dissemination . The lack of P-cad expression has been associated with a low OS rate in OSCC patients . In contrast, one study failed to detected an independent and significant association between E-cad expression and prognosis . Some transcriptional repressors have been shown to be important in the loss of E-cad expression by tumour cells. The expression of the transcriptional repressor SIP1, which regulates E-cad expression, has been correlated with a lower disease-specific OS in OSCC .

Catenins

The intercellular domain of E-cad binds to proteins known as catenins, forming the cadherin–catenin complex, which is involved in the intracellular transduction of cell-to-cell contact signals . U eda et al. showed that reduced expression of β- and γ-catenin can be used as a potential marker of poor OSCC prognosis.

Versican

Versican is one of the main components of the ECM. It is a chondroitin sulphate proteoglycan, a member of the aggrecan gene family, and has been associated with diverse interactions in a number of biological and pathological processes . Versican is overexpressed in diverse tumours, and it plays a main role in tumour growth by repressing cell adhesion, stimulating cell proliferation and migration, and regulating angiogenesis . It has been shown in a cohort of OSCC patients that high stromal versican expression is an independent predictor for an unfavourable prognosis .

Results

The search terms used for this review identified 22 different molecular biomarkers that had been investigated using IHC to assess prognostic parameters in OSCC patients. Table 1 demonstrates the prognostic relevance of all tumour biomarkers divided by group, as well as the variations in sample sizes and in P statistical values according to each specific marker studied amongst the different publications. The P values were obtained from multivariate Cox regression or Kaplan–Meier analysis (preferably overall survival) when the first was lacking. The biomarkers were classified into five groups based on their biological function: cell cycle acceleration and proliferation; tumour suppression and apoptosis; hypoxia; angiogenesis; and cell adhesion and matrix degradation.

Table 1
Prognostic relevance of 22 different biomarkers investigated through IHC in OSCC.
Molecular biomarker Prognostic significance Sample size and P value variation (min.-max.) a No prognostic significance Sample size and P value variation (min.-max.) a Total
Cell cycle acceleration and proliferation
EGFR 5 b 41–140 – 0.0002–0.02 2 47–165 – 0.114 7
Cyclin D1 2 b , c 140–348 – <0.0001–0.0181 2
Ki-67 3 41–113 – 0.0001–<0.001 1 74 – 0.091 4
PCNA 3 60 – 113 – >0.05 3
Akt1 1 84 – 0.013 1
Tumour suppression and apoptosis
p53 and p63 9 b 45–140 – 0.0002–0.049 9
p21 and p27 2 106–192 – 0.018–0.03 1 192 – 0.43 3
Bcl-2 family members 4 35–110 – <0.005–0.0489 1 110 – 0.4901–0.8287 5
pRb 2 220–348 – 0.004–<0.05 2
Survivin 1 78 – 0.002 1
Hypoxia
HIF-1α 3 57–112 – 0.004–0.048 3
CA IX 2 60–117 – 0.02–<0.05 1 68 – 0.062 3
GLUT-1 1 40 – 0.035 1
EPOR 1 43 – 0.030 1
Angiogenesis
VEGF 4 59–1002 – <0.001–0.045 1 84 – 0.168 5
CD105 3 26–176 – <0.001–0.02 3
Ephs 1 59 – 0.037 1
Cell adhesion and matrix degradation
MMPs 6 68–138 – 0.008–0.032 1 38 – data not shown 7
CD44 3 36–138 – 0.01–0.03 3
Cadherins 3 47–67 – <0.0001–<0.05 1 84 – data not shown 4
Catenins 1 135 – 0.0088 1
Versican 1 139 – 0.048 1
Total 58 12 70
Only gold members can continue reading. Log In or Register to continue

Feb 7, 2018 | Posted by in Oral and Maxillofacial Surgery | Comments Off on Prognostic significance of immunohistochemical biomarkers in oral squamous cell carcinoma
Premium Wordpress Themes by UFO Themes