Podoplanin expression in the development and progression of laryngeal squamous cell carcinomas

Head and neck squamous cell carcinoma (HNSCC) is the fifth most common cancer worldwide. Although in most studies HNSCC is considered as a single type of carcinoma, important differences in the clinical and biological behaviour have been observed depending on the location of the tumours (i. e. oral cavity, oropharynx, hypopharynx and larynx). Patients with HNSCC have benefited greatly from the latest advances in surgical techniques, radiation therapy and chemotherapy. However, despite the advancements in local control and overall quality-of-life achieved with the use of combined modality therapies, the survival rates for HNSCC have not improved significantly over the past two decades [1]. Hence, novel methods of cancer detection and prognostication need to be developed. Recent advances in genomic and basic research have increased our understanding of the molecular processes governing tumour formation and progression. HNSCC is a heterogeneous disease involving dysregulation of multiple pathways linked to cellular differentiation, cell cycle control, apoptosis, angiogenesis, and metastasis [2]. Thus, much work is focused on the identification of better biologic and molecular factors that may serve as prognostic and predictive markers [3].

Human podoplanin is a 38-kDa mucin-type transmembrane glycoprotein consisting of 162 amino acids. In normal tissues, podoplanin is expressed in kidney podocytes [4], skeletal muscle, placenta, lung and heart [5], in myofibroblasts of the breast and salivary glands, in osteoblasts and mesothelial cells [6]. Occasionally, focal expression of podoplanin can be found in circumscribed areas of the basal layer of the human epidermis [7]. As podoplanin is expressed on lymphatic but not on blood vessel endothelium, it has been widely used as a specific marker for lymphatic endothelial cells and lymphangiogenesis in many species [4], including HNSCC [8]

The expression of podoplanin is up-regulated in a number of different human cancers, including squamous cell carcinoma of the oral cavity, the lung, the cervix, the oesophagus, and the skin, in dysgerminomas of the ovary and granulosa cell tumours, in mesothelioma, and in many tumours of the central nervous system (CNS) [5, 7, 9‐12]). In addition, recent experimental results have demonstrated that podoplanin mediates a pathway leading to collective cell migration and invasion in vivo and in vitro[12].

The expression of podoplanin in human cancers and its relationship with tumour invasion raises the possibility that podoplanin expression could be used as a biomarker for diagnosis and prognosis. Supporting this notion, podoplanin has been identified as a marker of malignant transformation and poor prognosis in oral cancer [13, 14]. Since HNSCC may behave differently depending on the tumour site, in this work we analysed podoplanin expression in a series of squamous cell carcinomas and premalignant lesions of the larynx to ascertain the role of podoplanin in both malignant transformation and tumour progression and its clinical significance in laryngeal cancer.

The physiological function of podoplanin is still unknown. Podoplanin-deficient mice die at birth owing to respiratory failure exhibiting a phenotype of dilated, malfunctioning lymphatic vessels and lymphoedema [16]. In addition, podoplanin can induce platelet aggregation in vitro[17]. In the pathological situations studied thus far, the mouse homologue of podoplanin (PA2.26, OTS-8) is induced in mouse skin during tissue regeneration after wounding and treatment with carcinogenic phorbol 12-myristate 13-acetate [18]. OTS-8 is also induced by 12-Otetradecanoylphorbol- 13-acetate in mouse osteoblastic cells and is constitutively expressed in oncogenic Ras-transformed cells [19]. Podoplanin has also been found to promote tumour cell invasion by inducing collective cell migration via the down-regulation of the activities of small Rho family GTPases [20]. These findings suggest a role of podoplanin in tissue development and repair as well as in carcinogenesis and malignant progression.

In this study we found that podoplanin is expressed in a high percentage of dysplastic lesions and squamous cell carcinomas of the larynx. This is not surprising since podoplanin expression is mainly detected in squamous cell cancers, CNS tumours and germinal neoplasia; in contrast, expression of podoplanin has not been found in the majority of adenocarcinomas, including lung, colon and prostate cancers [20]. Podoplanin expression has been previously explored in other head and neck squamous cell carcinomas, such as oral and hypopharyngeal carcinomas [13], as well as in oral premalignancies [14]. However, our study is the first to analyse podoplanin expression in both laryngeal premalignant lesions and laryngeal carcinomas and its relationship with clinico-pathological parameters and prognosis.

In oral leukoplakia, high podoplanin expression has been associated with an increased risk of progression to invasive cancer, suggesting that podoplanin could serve as a powerful biomarker to predict the risk for oral cancer development in patients with oral leukoplakia [14]. This evidence supports the importance of podoplanin in oral tumourigenesis and malignant transformation. Similarly, our findings indicate that podoplanin is also frequently abnormally expressed in the early stages of laryngeal tumourigenesis and patients carrying podoplanin-positive dysplastic lesions (scores 2-3) exhibit a higher incidence of laryngeal cancer than patients with negative expression (51% versus 30%), although these differences did not reach statistical significance (P = 0.071). Since podoplanin expression was not associated with the severity of dysplasia, this trend towards a poorer prognosis for patients with podoplanin-positive dysplasias suggests a role for podoplanin in the progression to laryngeal cancer. In this study cohort, the histology of the lesions does not have a significant role in assessing laryngeal cancer risk, and podoplanin seems a stronger predictor. The unexpected high proportion of mild dysplasias with progression to laryngeal carcinoma in our cohort could be due to pure chance, given the limited number of patients with this diagnosis. However, this also underscores the limited value of histopathological classification in predicting outcome. These data suggest the utility of podoplanin as a biomarker for cancer risk assessment providing additional value beyond current clinical and histopathological evaluations.

Nevertheless, podoplanin expression alone may not be sufficient to promote tumourigenesis because many of the lesions (44%) exhibit protein expression only in the basal layer cells, and this was independent of the severity of the dysplasia. Other factors are therefore needed to promote clonal expansion of the abnormal cells. The upward clonal expansion of the podoplanin-expressing cells in the epithelial layers supports this notion. Indeed, lesions with such clonal expansion carry a higher risk of laryngeal cancer development. The ability to detect the cells expanding beyond basal layers may allow us to visualise potential clonal expansion, possibly from stem-cell clones, during tumourigenesis [14]. In fact, podoplanin has been identified as a marker of tumour-initiating cells (TICs) in squamous cell carcinomas [21]. Tumourigenicity and capability of recapitulating human SCC are by definition properties of TICs. Premalignant lesions with podoplanin expression beyond the basal cell layer may represent truly early neoplastic lesions, enriched in TICs and with a higher risk of progression to invasive cancer.

In relation to the possible role of podoplanin in tumour progression, it has been reported that high podoplanin expression significantly associates with nodal metastasis and reduced survival in oral squamous cell carcinomas [13]. In marked contrast to these findings, we did not find a significant association of podoplanin expression with lymph node metastasis nor poor prognosis in laryngeal cancer. On the contrary, patients with high podoplanin expression showed a better disease-specific survival. This could be explained by the fact that podoplanin expression was higher in early-stage tumours and, interestingly, all cases that developed distant metastasis showed low podoplanin expression. Indeed, we found a significant inverse association of podoplanin expression with disease stage and T classification. Podoplanin expression was also significantly more frequent in glottic tumours, which are generally smaller and detected at an earlier stage than supraglottic tumours. Our results showing that podoplanin expression levels decreased with primary tumour size evidence a higher proportion of podoplanin-positive cells in small tumours and podoplanin-positive staining in those tumours could reflect a higher proportion of TICs, rather than a higher invasive potential. In addition, podoplanin expression was also significantly higher in well-differentiated tumours, which are usually less invasive than those poorly differentiated. We also observed a trend towards better disease-specific survival for patients with a focal expression of podoplanin in the periphery of tumour nests, defined in some works as the invasive edge of the tumours [20]. There are contradictory data regarding the clinical significance and biological role of podoplanin expression in squamous cancers, with contrasting results depending on the tumour sites studied. Thus, in squamous cell carcinoma of the uterine cervix, low levels of podoplanin were significantly associated with the presence of lymphatic invasion and lymph node metastasis, as well as with shorter survival and higher risk of tumour recurrence [22, 23]. In addition, tumour emboli within lymphatic spaces as well as metastatic cells from lymph nodes showed no podoplanin immunostaining in the vast majority of tumours, even in those cases with positive expression in the main tumour mass [23]. In squamous cell carcinomas of the lung [24], patients who had podoplanin-positive tumours, and especially those with focal expression in the periphery of tumour nests (named hierarchical distribution pattern by the authors) showed a significantly better overall survival than those with podoplanin-negative tumours. In addition, podoplanin expression in these tumours inversely correlated with lymphatic invasion and lymph node metastasis. This is in marked contrast to the observations made by Yuan et al[13] in oral squamous cell carcinomas, although in good agreement with the results presented herein. The results presented by Shimada et al[24]and our results suggest that squamous cell carcinomas with focal expression of podoplanin in the periphery of tumour nests (which is equivalent to the hierarchical distribution pattern) may indicate lower biological aggressiveness. According to this, it is plausible that squamous cell carcinomas showing focal expression pattern represent a well-organised tumour group based on the TICs concept, whereas squamous cell carcinomas with a diffuse expression pattern could reflect disordered tumours in terms of the developmental hierarchy.

Hence the role of podoplanin in tumour initiation and progression remains elusive. Its involvement in tumour metastasis, however, has been demonstrated in an experimental model to be due to its platelet aggregation-inducing activity leading to pulmonary retention of CHO cells that overexpress podoplanin [25]. It has also been demonstrated that podoplanin contributes to tumour invasion by binding ERM proteins to activate RhoA resulting in epithelial-mesenchymal transition [26]. Although podoplanin-positive TICs in squamous cell carcinomas may use these mechanisms to initiate and sustain tumour growth, they may also proliferate rapidly through the activation of the SHH signalling pathway [21]. In addition to these intrinsic mechanisms, the microenvironment also influences the ability of TICs to generate tumours [21]. Considering their localization, it has been proposed that TICs may be regulated by stromal cells, which is comparable to the regulation of stem cells by their environmental niche [21]. Histologically, podoplanin-positive cells were specifically located in most cases at the basal region of squamous cell carcinoma tumour nests, close to the surrounding stromal cells and the tumour-microenvironment interaction plays a decisive role in cancer progression. It has been recently described that positive podoplanin expression in stromal fibroblasts exerts a protective role against cell invasion and is a significant indicator of good prognosis in patients with advanced colorectal cancer [27].

We have demonstrated for the first time that podoplanin is expressed in a high percentage of laryngeal dysplasias and laryngeal squamous cell carcinomas. Podoplanin-positive dysplasias had a higher risk of progression to invasive carcinoma than those with negative expression, although the differences did not reach statistical significance. Prospective studies involving larger numbers of patients are needed to further evaluate the clinical utility of podoplanin as a biomarker for laryngeal cancer risk assessment providing additional value beyond the clinical and histological markers. Podoplanin expression in laryngeal squamous cell carcinomas, however, diminishes during tumour progression and does not correlate with invasive potential. Taken together, these data support a role for podoplanin expression in the initiation rather than in the progression of laryngeal cancers.