Molecular biomarkers of prognosis in laryngeal squamous cell carcinoma

Introduction Laryngeal squamous cell carcinoma represents an important cause of cancer­related death. Laryngeal squamous cell carcinoma is the sec­ ond most common type of head and neck malignancies, with a total of 12,000 new cases diagnosed yearly in the United States. The clinical value of several molecules as molecular biomarkers for prognosis of laryn­ geal squamous cell carcinoma as well as for monitoring the response of laryngeal squamous cell carci­ noma patients to treatment is high. This review summarises the clinical importance of tumour protein p53, proliferating cell nuclear antigen, marker of proliferation Ki­67, cyclins, cyclin­dependent kinase 4, inhibitors of cyclin­dependent kinases, epidermal growth factor receptor, vascular endothelial growth factor A and its receptor, B­cell CLL/lymphoma 2 (BCL2) protein family members, kal­ likrein­related peptidase 11, 3,4­di­ hydroxy­l­phenylalanine decarbox­ ylase and microRNAs. Conclusion Deregulation of protein or mRNA expression of these genes in laryn­ geal squamous cell carcinoma com­ pared to benign laryngeal tumours, dysplasias, or normal tissues of the larynx suggests that these potential molecular biomarkers merit further validation owing to their impor­ tant prognostic value in laryngeal squamous cell carcinoma treatment. Introduction Head and neck cancer is the sixth most commonly diagnosed cancer all over the world. The overwhelm­ ing majority of head and neck cases are characterised as squa­ mous cell carcinomas (SCCs). Head and neck squamous cell carcinoma (HNSCC), which represents an impor­ tant cause of cancer­related death, starts from squamous cells lining the moist, mucosal surfaces inside the mouth, the nose and the throat. Thus, such tumours can develop in nasal cavity, paranasal sinuses, oral cavity, larynx, trachea, hypolarynx, nasopha­ rynx, oropharynx, ears and salivary glands. Laryngeal squamous cell carcinoma (LSCC) is the second most common type of HNSCC, with a total of 12,000 new cases diagnosed yearly in the US. The incidence of LSCC is greater in men than in women, especially for people between the sixth or seventh decade of their life1. The causative risk factors of HNSCC include tobacco use and alcohol consumption as well as other habits that are likely to be implicated in laryngeal tumourigenesis. Deregulation of the expression of many genes is related to many hallmarks of cancer, such as uncon­ trolled cell proliferation, impaired apoptosis, progressive loss of cell differentiation (anaplasia), epithe­ lial–mesenchymal transition, meta­ stasis and angiogenesis. Besides that, the clinical value of such mole­ cules as molecular biomarkers for diagnosis, prognosis or monitoring of patient response to treatment is high. Molecular biomarkers are needed in clinical practice, to achieve individu­ alisation of anticancer treatment. In this context, immunohistochemi­ cal assessment and quantification of messenger RNA (mRNA) expression of emerging molecular biomarkers will assist utmost the generation of novel screening tests possessing high sensi­ tivity and specificity, as well as tailormade therapies counteracting LSCC. Moreover, selective impairment of interactions between key players of biochemical processes could signifi­ cantly decelerate tum our progression and, hence, elongate survival of LSCC patients. The aim of this review is to discuss the mole cular biomarkers of prognosis in laryngeal squamous cell carcinoma. Discussion The author has referenced some of his own studies in this review. These ref­ erenced studies have been conducted in accordance with the Declaration of Helsinki (1964) and the protocols of these studies have been approved by the relevant ethics committees rela­ ted to the institution in which they were performed. All human subjects, in these referenced studies, gave informed consent to participate in these studies. Tumour protein p53 Tumour protein p53, expressed by the TP53 gene, is a tum our­ suppressor protein that keeps many genes under transcriptional con­ trol in response to many different cellular stresses. p53 can induce cell cycle arrest, senescence, DNA repair and apoptosis. Although the potential associations of p53 with clinicopathological features of LSCC as well as the prognostic significance of p53 expression in LSCC have been intensively studied during the last 20 years; the resulting data are contradictory. Variations in the selection of LSCC patients


Introduction
Head and neck cancer is the sixth most commonly diagnosed cancer all over the world.The overwhelm ing majority of head and neck cases are characterised as squa mous cell carcinomas (SCCs).Head and neck squamous cell carcinoma (HNSCC), which represents an impor tant cause of cancerrelated death, starts from squamous cells lining the moist, mucosal surfaces inside the mouth, the nose and the throat.Thus, such tumours can develop in nasal cavity, paranasal sinuses, oral cavity, larynx, trachea, hypolarynx, nasopha rynx, oropharynx, ears and salivary glands.Laryngeal squamous cell carcinoma (LSCC) is the second most common type of HNSCC, with a total of 12,000 new cases diagnosed yearly in the US.The incidence of LSCC is greater in men than in women, especially for people between the sixth or seventh decade of their life 1 .The causative risk factors of HNSCC include tobacco use and alcohol consumption as well as other habits that are likely to be implicated in laryngeal tumourigenesis.
Deregulation of the expression of many genes is related to many hallmarks of cancer, such as uncon trolled cell proliferation, impaired apoptosis, progressive loss of cell differentiation (anaplasia), epithe lial-mesenchymal transition, meta stasis and angiogenesis.Besides that, the clinical value of such mole cules as molecular biomarkers for diagnosis, prognosis or monitoring of patient response to treatment is high.Molecular biomarkers are needed in clinical practice, to achieve individu alisation of anticancer treatment.In this context, immunohistochemi cal assessment and quantification of messenger RNA (mRNA) expression of emerging molecular biomarkers will assist utmost the generation of novel screening tests possessing high sensi tivity and specificity, as well as tailormade therapies counteracting LSCC.Moreover, selective impairment of interactions between key players of biochemical processes could signifi cantly decelerate tum our progression and, hence, elongate survival of LSCC patients.The aim of this review is to discuss the mole cular biomarkers of prognosis in laryngeal squamous cell carcinoma.

Discussion
The author has referenced some of his own studies in this review.These ref erenced studies have been conducted in accordance with the Declaration of Helsinki (1964) and the protocols of these studies have been approved by the relevant ethics committees rela ted to the institution in which they were performed.All human subjects, in these referenced studies, gave informed consent to participate in these studies.

Tumour protein p53
Tumour protein p53, expressed by the TP53 gene, is a tum our suppressor protein that keeps many genes under transcriptional con trol in response to many different cellular stresses.participating in each study are likely to account for this discrepancy.In addition, there has been much con troversy about the capacity of p53 to predict radiotherapy failure and locoregional recurrence in LSCC patients.
p53 overexpression does not seem to be associated with biological fea tures of LSCC.In more detail, no significant associations have been found between immunohistochemi cally detected p53 expression and depth of tumour invasion, histologi cal grade, status of regional lymph nodes, or TNM stage of LSCC patients as well as with progression of the disease 2 .Although Hirvikoski et al. 3 has proposed a favourable prognostic role for strong p53 immunostaining in LSCC, numerous studies showed that p53 is a rather unfavourable predictor of diseasefree survival (DFS) and overall survival (OS) [3][4][5] .However, many other studies failed to demonstrate any prognostic significance for this tumoursuppressor protein.Hence, p53 immunopositivity could not predict tumour recurrence of radiotherapytreated patients suffering from glottic LSCC 2 .Conse quently, larger studies are needed to examine p53 expression in laryngeal tumours of patients subjected to different treatment modalities and to assess its prognostic importance.

Proliferating cell nuclear antigen
Proliferating cell nuclear antigen (PCNA) is a cofactor of DNA poly merase delta.PCNA expression in paraffin-embedded tissue sections is indicative of the cancer cell pro liferation and is associated with histological tumour differentiation, vascular and lymphatic invasion, depth of tumour invasion, nodal status, presence of neck metastases and locoregional recurrence.PCNA expression in preoperative biop sies has been proposed as a reliable predictor of presence of occult neck metastases 6 .PCNA overexpression is also associated with low DFS and OS rates 7 .Thus, immunohistochemical assessment of PCNA expression in LSCC could prove a valuable tool for determination of prognosis and selec tion of the appropriate therapeutic treatment.

Marker of proliferation Ki-67
Marker of proliferation Ki67 (MKI67) is a nuclear protein, inv olved in ribo somal RNA (rRNA) synthesis and cell proliferation.MKI67 overexpression was associated with high histologic grade.MKI67 expression was also elevated in nodepositive LSCC patients, compared to patients with out local metastases 6 .Furthermore, LSCC patients with high MKI67 expres sion in their tumour recurred loco regionally more frequently if treated with splitcourse radiotherapy than if treated with a continuous course of therapy 8 .MKI67 overexpression can also predict the presence of occult neck metastases in LSCC 6 .As a result, immunohistochemical assessment of MKI67 expression could select those patients who are more likely to benefit from elective neck dissection.

Cyclins, cyclin-dependent kinases and their inhibitors
Cyclins are members of a highly con served protein family, which control the cell cycle.They form complexes with cyclindependent kinases and hence activate them.These cellcycle regulators exhibit an important peri odicity through the different phases of the cell cycle.The most studied cyc lins in LSCC are cyclin A2 (CCNA2; in the past, known as cyclin A), cyclin B1 (CCNB1), cyclin D1 (CCND1), cyclin D3 (CCND3) and cyclin E1 (CCNE1; in the past, known as cyclin E).
Protein expression levels of CCNB1, CCND1 and CCNE1 are related to the site of the tumour, depth of tumour invasion and stage of the disease.In more detail, CCNB1, CCND1 and CCNE1 immunopositivities are associated with the existence of LSCC located in the supraglottic larynx, highly invasive (T3 and T4) malignant neoplasms of the larynx and tumours being at an advanced clinical stage (III and IV) 7,9,10 .More over, overexpression of CCND1 and CCNE1 was linked to positive nodal status 7,9 .Moreover, the CCNE1 gene was overexpressed in grade III LSCC, compared to grades I and II LSCC 7 .Besides CCND1 protein over expression, high CCND1 mRNA expression was observed in several LSCC specimens and was associated with local invasion and stage IV LSCC.It has been suggested that CCND1 gene amplification could acc ount for high CCND1 mRNA levels that were measured in several LSCC specimens.It should also be noted that CCND1 gene amplification was associated with the presence of metastases in regional lymph nodes 11 .
Cyclins also possess a significant prognostic potential in LSCC.CCNA2 immunopositivity was indicative of locoregional recurrence in LSCC, as the risk of locoregional relapse within a 5year interval from the time of LSCC diagnosis was double for patients with CCNA2positive LSCC than for those with CCNA2negative laryngeal tumours.Overexpression of CCNA2 also predicted poor DFS and OS in LSCC patients who had been treated with surgery and post operative radiotherapy 8 .In addition, CCNB1 immunopositivity was shown to constitute an unfavourable-though not independent-prognosticator in this malignancy 10 .The most signifi cant unfavourable predictors of DFS and OS were CCND1 and CCNE1 7,9,12 .In addition to these cyc lins, high CCND3 mRNA exp ression was an independent prognosticator of poor outcome in LSCC 13 .
The coexpression of CCND1 and CCND3 in laryngeal cancerous tissue specimens was a very strong and reliable prognosticator, as patients with CCND1+/CCND3+ tumours had smaller OS rates than all the other LSCC patients, and patients with highest OS probabilities, whereas patients with CCND1−/CCND3+ or CCND1+/CCND3− LSCC had an inter mediate prognosis 13 .Furthermore, coexpression of CCND1 and CDK4 predicted poor patient outcome in the total of LSCC patients, but also in the subgroup of earlystage (I and II) LSCC cases 9 , similar to CCNE1 over expression 7 .Consequently, it appears that assessment of expression of cyclins could contribute to the appro priate management of LSCC patients by indicating those with poor prog nosis and those who should be treated more aggressively.
The enzymatic activity of activated cyclindependent kinases can be inhi bited by specific proteins, usually during the G1 phase of the cell cycle or in response to environmental stress or damaged DNA.The most representative examples of such proteins are cyclindependent kinase inhibitors, such as cyclindependent kinase inhibitor 1A (CDKN1A; also known as p21, CIP1 and WAF1), cyclindependent kinase inhibitor 1B (CDKN1B; also known as p27 and KIP1), cyclindependent kinase inhibitor 2A (CDKN2A; also known as p21, p16, p14, INK4A and ARF), and cyclindependent kinase inhi bitor 2B (CDKN2B; also known as p15 and INK4B).
CDKN1A and CDKN1B immunore activities were related to clinicopath ological characteristics of malignant laryngeal tumours.In particular, low CDKN1A expression was associ ated with grade III tumours and pres ence of metastases in regional lymph nodes in LSCC patients.Moreover, weak CDKN1B staining was associa ted with high depth of tumour inva sion (T3 and T4) and advanced clinical stage (stages III and IV) 12 .As expected, LSCC patients with DKN1Apositive tumours had longer OS intervals than those with CDKN1Anegative neoplasms 14 .CDKN1B expression is another indep endent favourable prognosis indicator in LSCC, as it predicts better DFS and OS rates.
On the other hand, patients with CDKN1B−/CCND3+ LSCC had a very poor prognosis, in terms of DFS and OS 12 .In basaloid LSCC-a rather uncommon but aggressive variant of LSCC-loss of CDKN1B expression was associated with high tumour aggressiveness and poor clinical outcome 15 .Moreover, deletion of CDKN2A and/or CDKN2B genes was associated with disease progres sion and poor OS in LSCC patients 16 .In general, loss of expression of cyclindependent kinase inhibitors predicts unfavourable prognosis in LSCC.

Epidermal growth factor receptor
The epidermal growth factor recep tor (EGFR) is a transmembrane receptor exhibiting tyrosine kinase activity.EGFR is overexpressed in LSCC, in comparison with normal laryngeal tissue specimens.It has recently been suggested that EGFR expression could identify LSCC patients at high risk for locoregional recurrence.Overexpression of the EGFR gene at the protein level was also observed in poorlydifferentiated LSCC and predicted an increased risk of reg ional metastatic recurrence 17 .EGFR immunopositivity was dem onstrated to predict shortterm relapse and poor outcome in LSCC patients, independently of other significant prognostic factors includ ing the tumour extent, nodal status and histological grade 18 .Bes ides its high significance as a mole cular prognostic biomarker, EGFR expres sion probably constitutes a predictive biomarker for the identification of LSCC patients who are most likely to benefit from accelerated radiotherapy with carbogen (98% O 2 ; 2% CO 2 ) and nicotinamide (ARCON), a therapeutic approach aiming at high loco regional control, in particular for LSCC with low EGFR levels 19 .Thus, positive EGFR expression detected at the time of diagnosis could assist decision making concerning LSCC treatment.

Vascular endothelial growth factor A and its receptor
The vascular endothelial growth factor A (VEGFA) is produced by cancer and stroma cells and exerts its angiogenic action by binding to its receptors, one of which is fms related tyrosine kinase 1 (FLT1) 20 .VEGFA levels in serum were signi ficantly higher in patients with advancedstage LSCC than in healthy controls, and this overexpression predicted a more aggressive LSCC phenotype and a worse clinical outcome 21 .VEGFA expression was significantly associated with tumour size, histological grade and presence of metastases in regional lymph nodes of LSCC patients.Furthermore, VEGFA and FLT1 mRNA expression was associated with high depth of tumour invasion and predicted independently shortterm relapse of LSCC patients 20 .In addition, strong VEGFA immunostaining was an inde pendent unfavourable prognosticator of OS 22 .

BCL2 family members
BCL2 family proteins promote or inhibit apoptosis and are, hence, either antiapoptotic (e.g.BCL2) or proapoptotic members (e.g.BAX).Nonetheless, some BCL2 family genes encode both antiapoptotic and proapoptotic protein isoforms.Expression of the antiapoptotic BCL2 protein is associated with positive nodal status and advanced TNM stage 23 .Moreover, mRNA expres sion of another member of the BCL2 family, BCL2L12, was shown to be downregulated in advancedstage LSCC 24 .BCL2 immunopositivity, either alone or in combination with BCLX L expression and reduced BAX levels predicts radiotherapy failure, thus implying that inhibition of apoptosis accounts for radiotherapy resistance 25 .the tissue kallikrein and kallikrein related peptidases.KLK11 mRNA exp ression was found to be drama tically lower in LSCC of primary or recu rrent origin, in comparison with adjacent non cancerous laryngeal tissue speci mens.In addition, KLK11 mRNA positivity was more common among earlystage (I and II) carcinomas of the larynx than among laryngeal tumours of advanced TNM stage (III and IV).Patients with KLK11 mRNA positive LSCC exhibited prolonged OS interval compared to those with KLK11 mRNAnegative LSCC 26 .Thus, KLK11 is likely to constitute a pro mising diagnostic and/or prognostic biomarker in this malignancy.l-DOPA decarboxylase lDOPA decarboxylase (DDC) is a pyridoxalphosphatedependent enzyme catalysing the decarboxyl ation of 3,4dihydroxylphenylala nine (lDOPA) to dopamine as well as the decarboxylation of 5hydroxy ltryptophan (5HTP) to serotonin.Quantitative mRNA expression analysis of the DDC gene in primary tumours of the larynx and their non cancerous counterparts revealed its upregulation in LSCC.DDC mRNA expression was shown to constitute a potential diagnostic biomarker, which merits further evaluation in large cohorts of laryngeal tissue samples.Moreover, DDC mRNA expression status is inversely associated with progression of LSCC, as earlystage (I and II) tumours exhibit higher DDC mRNA levels than advancedstage (III and IV) tumours 27 , suggesting that DDC mRNA expression could repre sent a novel mole cular prognostic biomarker in LSCC.microRNAs microRNAs (miRNAs) are small noncoding RNAs of approxi mately 19-25 nucleotides that post transcriptionally regulate finely proteincoding gene expression.miRNAs bind to the 3′-untranslated region of targeted mRNAs and trigger translational repression and, some times, mRNA degradation.miRNAs are heavily involved in laryngeal tumourigenesis and represent very promising biomarkers in LSCC as well as in other malignancies, as they can be accurately quantified in FFPE samples and blood plasma or serum of patients, owing to their stability.

Kallikrein
Recently, a panel of 26 miRNAs being differentially expressed in the blood plasma of LSCC patients compared to the control cohort has been discovered.Of these, 16 miRNAs were significantly upregu lated and 10 miRNAs were signifi cantly downregulated in the LSCC patients' blood plasma.Most impor tantly, 17 miRNAs were shown to exist only in the blood plasma of LSCC patients 28 .Thus, this LSCC-specific miRNA signature could serve as a noninvasive molecular biomarker for LSCC.Besides them, three other miRNAs (miR155, miR4555p and miR196a) were demonstrated to be overexpressed in laryngeal tumours, compared to their noncancerous counterparts.High levels of miR155, miR4555p and miR196a were cor related with the progression of the disease.In particular, there was a progressive increase of miR4555p and miR196a levels during laryn geal tumourigenesis, being very low in normal laryngeal tissues, intermediate in benign tumours, higher in dysplasias and very high in mali gnant tumours of the larynx.miR196a and miR155 expression was more elevated in highly invasive (T3 and T4) LSCC than in low invasive (T1 and T2) tumours 29,30 .miR155 overexpression was also ass ociated with poorly differenti ated (grade III) LSCC 29 .Interestingly, several miRNAs among those which are differentially expressed in LSCC patients could be combined in a multifactorial panel of biomarkers with diagnostic and/or prognostic purposes.

Conclusion
The clinical significance of all these potential diagnostic, prognostic and treatmentresponse biomarkers in LSCC has been intensively investigated so far.Deciphering of the signalling pathways that are implicated in LSCC progression and metastasis is likely to contribute utmost to the discovery of novel molecular biomarkers that could be applied in clinical practice.Deregulation of protein and/or mRNA expression in LSCC compared to benign laryngeal tumours, dysplasias, or normal laryngeal tissues suggests further validation for these potential biomarkers in larger coh orts of LSCC patients.

Abbreviations list
p53 can induce cell cycle arrest, senescence, DNA repair and apoptosis.Although the potential associations of p53 with clinicopathological features of LSCC as well as the prognostic significance of p53 expression in LSCC have been intensively studied during the last 20 years; the resulting data are contradictory.Variations in the selection of LSCC patients Licensee OA Publishing London 2013.Creative Commons Attribution License (CC-BY) For citation purposes: Kontos CK.Molecular biomarkers of prognosis in laryngeal squamous cell carcinoma.OA Cancer 2013 Sep 01;1(2):12.
-related peptidase 11 Kallikreinrelated peptidase 11 (KLK11) is a trypsinlike serine pep tidase.It is a member of the family of Licensee OA Publishing London 2013.Creative Commons Attribution License (CC-BY) For citation purposes: Kontos CK.Molecular biomarkers of prognosis in laryngeal squamous cell carcinoma.OA Cancer 2013 Sep 01;1(2):12.