Elsevier

Clinical Biochemistry

Volume 45, Issue 9, June 2012, Pages 623-630
Clinical Biochemistry

Diagnostic and prognostic significance of human kallikrein 11 (KLK11) mRNA expression levels in patients with laryngeal cancer

https://doi.org/10.1016/j.clinbiochem.2012.03.005Get rights and content

Abstract

Objectives

Human kallikrein 11 gene (KLK11) encodes a secreted serine protease. In view of its diagnostic and prognostic strength in many malignancies, we investigated the mRNA expression levels of KLK11 in laryngeal tissues in order to unveil its clinical usefulness in laryngeal cancer.

Design and methods

KLK11 expression was quantified in 163 tissue samples from 105 laryngeal cancer patients with the development of a highly sensitive real-time PCR methodology, using SYBR Green® chemistry.

Results

KLK11 expression in laryngeal cancer specimens of primary or recurrent nature was significantly inferior compared with their non-malignant counterparts (P < 0.001 and P = 0.026, respectively), a finding of immense diagnostic value as illustrated in the ROC curve analyses (P < 0.001). Survival analysis showed that patients harboring KLK11-positive tumors had a significantly decreased risk of death (HR = 0.26, P = 0.042).

Conclusions

Our data recommend KLK11 mRNA expression as a novel and independent biomarker in laryngeal cancer for diagnostic and prognostic purposes.

Highlights

► We examined KLK11 mRNA expression in cancerous and non-cancerous laryngeal tissues. ► KLK11 was downregulated in laryngeal cancer, a finding of diagnostic importance. ► High KLK11 expression was associated with early-stage disease and favorable prognosis. ► KLK11 could serve as a diagnostic and prognostic indicator for laryngeal cancer.

Introduction

Cancer of the larynx comprises the most frequent form of neoplasia in the wider anatomic region of head and neck, accounting for approximately 2% of all newly diagnosed malignancies around the globe [1]. The grand majority of these tumors are categorized as laryngeal squamous cell carcinomas (LSCC), developing via malignant transformation of the mucosal epithelial cells which cover the upper respiratory tract [2]. The incidence of LSCC is increased in men, especially those found between the 6th and 7th decade of life, with the male/female ratio calculated to be much higher than in other malignancies of the head and neck area [2], [3]. Among the causative risk factors of the disease, tobacco use and alcohol consumption hold a position of principal importance in the carcinogenesis of the larynx, since a percentage greater than 95% of the patients suffering from LSCC has a medical history of tobacco smoking and/or alcohol abuse before diagnosis [4].

Over the past 30 years, a remarkable progress has been recorded both in diagnosis and treatment of LSCC, mainly attributed to the application of combined therapy regimens and novel surgical approaches aiming at preserving the organ and its vital functions. Despite the above mentioned improvements in the quality of life, the overall survival rate for patients with cancer of the larynx remains stable and among the lowest in comparison to other types of cancer [5], [6]. The striking heterogeneity of the biological behavior of LSCC resulting from the distinct molecular and genetic background of each malignant tumor, the occasional inadequacy of the TNM classification and the scarcity of reliable and informative prognosticators constitute the most likely causes of this unusual high cancer-related mortality rate [5], [7].

Further developments in unraveling and understanding the fundamental molecular pathways governing the neoplastic transformation and progression towards LSCC led to the identification of new potential molecular markers for the disease with both diagnostic and prognostic roles [7], [8]. However, none of those biomarkers has been yet established in the clinical practice, because they lack specificity and/or their diagnostic and prognostic significance has not been sufficiently evaluated. Up to date, the solely considered prognostic factors for the management of this type of malignancy are the TNM stage and the presence of nodal metastasis [5], [7], [9]. Therefore, it is peremptory to identify and implement novel tissue biomarkers, characterized by great sensitivity and specificity, so as to effectively contribute in the fields of diagnosis, prognosis and therapy of laryngeal tumors.

Human tissue kallikreins (KLKs) form an evolutionary conserved subgroup of secreted serine proteases that consists of fifteen structurally homologous genes located at the long arm of chromosome 19 [10]. KLKs not only share a large number of molecular features at both the gene and protein level, but also they show similarities as far as their expression patterns and regulatory mechanisms are concerned. These secreted endoproteases are co-expressed in a variety of tissues and biological fluids, where they exert their trypsin or chymotrypsin-like activity by catalyzing a broad spectrum of physiological processes, acting either as individual enzymes or through complex proteolytic cascades [11], [12]. Nevertheless, the human kallikrein family has gained attention in the last few years as soon as the clinical applicability of some of its members as cancer biomarkers has been brought to the limelight. Countless studies have revealed that the dysregulated mRNA and/or protein expression profiles of almost all human kallikreins in several types of malignancy are often in accordance with tumor-related characteristics, providing in that way invaluable prognostic information [13].

Human kallikrein 11 (KLK11), formerly known as trypsin-like serine protease (TLSP) and hippostasin, was originally cloned from human hippocampus cDNA via PCR. Few years later, this gene was integrated in the kallikrein multigene family, constituting one of its newest members [10], [14]. KLK11 resides at the chromosomal region 19q13.3–q13.4, surrounded by the KLK10 and KLK12 genes, and consists of six exons, the first of which is non-coding, and five intervening introns [15]. The presence of alternative transcripts is a very usual phenomenon between the members of the KLK family and so far three different tissue-specific splice variants of KLK11 have been recognized designated as isoform 1 (brain type), isoform 2 (prostate type) and isoform 3. These three protein isoforms, though varying in size, are secreted and become functional in the extracellular environment, since they contain the preserved amino acid residues of the catalytic triad of the enzyme's active center [16], [17], [18].

The protein is produced as a preproenzyme from which the secreted mature enzyme with trypsin-like substrate specificity is generated, after the appropriate proteolytic processing [19]. KLK11 mRNA expression has been demonstrated in several normal tissues, including prostate, stomach, trachea and skin, while its protein product has been detected in various biological fluids, such as seminal plasma, amniotic fluid and serum [20]. Even though KLK11 expression profile is quite extensive, its physiologic substrates are far from being delineated [19]. The diagnostic and prognostic potential of KLK11 gene expression levels has been reported mostly in endocrine-related cancers, such as those of the prostate gland and ovaries [13].

The purpose of the current study was the quantitative expression analysis of KLK11 in laryngeal tumors, by exploiting the advantages of the robust and sensitive real-time PCR methodology developed and the clinical evaluation of its possible diagnostic and prognostic significance in cancer of the larynx.

Section snippets

Laryngeal cancer patients and tissue samples

This retrospective study included 163 tissue specimens derived from 105 patients, who were diagnosed with laryngeal cancer and had undergone surgical excision of their LSCCs at Hippokration Hospital, Athens, Greece, during the period 2005–2010. Of the 163 tissue samples obtained, 105 were of malignant origin, 83 of which corresponded to primary LSCC, while the remaining 22 were acquired from patients with local recurrence in the larynx. Furthermore, matched adjacent non-neoplastic laryngeal

Quantitative analysis of KLK11 expression in laryngeal tissues with qPCR

The measurement of KLK11 expression levels in the entire set of laryngeal tissue specimens was accomplished by developing and optimizing a real-time PCR method of explicit sensitivity and specificity with the utilization of SYBR® Green I as the reporter dye of the reaction. The comparative Ct method (2 ΔΔCt) enabled us to quantify the mRNA expression levels of KLK11, by exploiting the derivable Ct values provided from the amplification plots of GAPDH and KLK11 (Fig. 1A). In order for this

Discussion

Laryngeal cancer, representing the eleventh most usual neoplasia affecting men in Greece with an incidence of 4.2%, remains a considerable source of morbidity and mortality, despite attempts to improve patients’ survival through means of precise stratification and efficacious therapeutic interventions [7], [23]. For this reason, the discovery of biomarkers, covering various aspects of the multistep course of malignant transformation in the larynx, may serve as an additional tool to combat this

Conflict of interest statement

All authors declare no conflicts of interest.

Acknowledgments

This project is part of the MSc thesis conducted by Christos Patsis during the postgraduate course “Clinical Biochemistry-Molecular Diagnostics”, which was supported financially by a scholarship from Bodossaki Foundation. We would, also, like to thank Bodossaki Foundation for the generous donation of the thermal cycler ABI Prism 7500.

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