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10.1245/s10434-006-9201-y
Annals of Surgical Oncology 14:248-257 (2007)
© 2007 Society of Surgical Oncology
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Original Article

Intratumoral Lymphatic Vessels and VEGF-C Expression Are Predictive Factors of Lymph Node Relapse in T1–T4 N0 Laryngopharyngeal Squamous Cell Carcinoma

Adolfo Hinojar-Gutiérrez1, María-Encarnación Fernández-Contreras2, Rocío González-González3, María-Jesús Fernández-Luque3, Adolfo Hinojar-Arzadún1, Miguel Quintanilla4 and Carlos Gamallo2

1 Department of Otorhynolaryngology, Hospital Universitario de la Princesa, Universidad Autónoma de Madrid (UAM), Madrid, Spain
2 Department of Pathology, Hospital Universitario de la Princesa, Universidad Autónoma de Madrid (UAM), Madrid, Spain
3 Department of Methodological Support Unit of the Foundation for Biomedical Research, Hospital Universitario de la Princesa, Universidad Autónoma de Madrid (UAM), Madrid, Spain
4 Department of Cell Signaling. Instituto de Investigaciones Biomédicas "Alberto Sols", Universidad Autónoma de Madrid (UAM), Madrid, Spain

Correspondence: Address correspondence and reprint requests to: Carlos Gamallo, Servicio de Anatomía Patológica, Hospital Universitario de la Princesa, 2a Planta, C/Diego de León, 62, 28006, Madrid, Spain; E-mail: cgamallo.hlpr{at}salud.madrid.org


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: The presence of intratumoral lymphatic vessels (ILVs) and the expression of vascular endothelial growth factor-C (VEGF-C) in tumour cells have been studied as markers of lymphangiogenesis in order to evaluate their role in metastatic dissemination in laryngopharyngeal squamous cell carcinoma.

Methods: A retrospective study was performed in 76 patients of N0 laryngopharyngeal carcinoma. with variable tumour size (T1–T4), histological grade, and location (supraglottic, glottic and hypopharyngeal). The presence of ILVs, as revealed by the expression of PA2.26 antigen and VEGF-C expression, were determined by immunohistochemistry (IHC). Low-grade and high-grade lymphangiogenesis were defined by qualitative and quantitative criteria.

Results: Multivariate analysis revealed low-grade ILV and VEGF-C expression to be associated respectively with 30.3- and 16.2-fold higher probabilities of cervical lymph node relapse (P = 0.005 and P = 0.032) and with 16.2- and 8.44-fold shorter disease-free survival (P = 0.009 and P = 0.045).

Conclusions: Low-grade ILV and VEGF-C expression are independent predictive factors of cervical lymph node relapse and shortening of time to relapse in N0 laryngopharyngeal carcinoma.

Key Words: VEGF-C • Lymphangiogenesis • Intratumoral lymphatic vessels • Larynx


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In Spain, the incidence and mortality rates of larynx cancer in male patients are the highest in the European Union.1 The occurrence of regional lymph node metastases is an essential prognostic factor in human tumours and forms the basis for various therapeutic models.2,3

Most patients (about 70–80%) with N0 laryngeal cancer will not develop metastasis in their cervical lymph nodes.4 Thus, the identification of those patients at higher risk of cervical lymph node relapse is essential for an accurate choice of the most suitable approach to healing the disease while avoiding overtreatment.

In recent years, several research groups have given us a better understanding of the processes of tumor growth and metastasis, and provided new tools to help identify those tumors with a higher probability of metastasis.5,6 The discovery of markers specific to lymphatic endothelium has allowed the study of lymphatic microcirculation, which in conjunction with the analysis of endothelial growth factors, has focussed attention on the tumor lymphangiogenesis process and its involvement in the evolution of the disease.7

The aim of this study was to analyze the intratumoral lymphatic vessels (ILVs), and the expression of the lymphangiogenic vascular endothelial growth factor-C (VEGF-C) in laryngopharyngeal squamous cell carcinoma. The association between the two variables, their relationship with clinicopathological factors typically linked with laryngopharyngeal carcinoma, the nature of lymph node relapse and their pattern of survival are also investigated.


   PATIENTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients and samples
Seventy-six patients (74 males and 2 females) who underwent surgery for N0 laryngopharyngeal squamous cell carcinoma, without prior treatment and negative surgical borders, were retrospectively studied. Their clinicopathological characteristics are summarized in Table 1Go. They were collected from the archives of the Otorhinolaryngology Department of the Hospital Universitario de la Princesa, Universidad Autónoma de Madrid (UAM). The median age of patients was 64 years (range, 41–87 years), and their median clinical follow-up period was 71 months (range, 40–128). On the basis of their tumour size, subjects were grouped as early (T1–T2; 48%) or advanced (T3–T4; 51.7%). Tumour staging was performed according to the American Joint Committee on Cancer/International Union Against Cancer (AJCC/UICC) 6th edition TNM staging system.8 Information about tumor histological grade was available from 69 patients. Only 11 patients received post-surgical treatment, consisting of radiotherapy in all cases, and the remaining 65 patients received surgery alone. Thirty-two patients did not undergo cervical dissection, 29 of them featuring early glottic stages (Table 1Go). All patients, except those with T1a glottic tumours were subjected to cervical computed tomography (CT).


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  		TABLE 1. Characteristics of patients studied
 
Immunohistochemistry
Samples were paraffin-embedded surgical specimens collected from the Archives of the Department of Pathology of the Hospital Universitario de la Princesa (UAM). All specimens had been fixed in 10% formalin. For study purposes, the most representative sections of the tumour were selected, together with a control of normal tissue.

Initial intratumoral lymphatic vessel evaluation by the analysis of PA2.26 antigen expression
Intratumoral lymphatic vessels (ILVs) were identified initially. Matched samples of healthy laryngeal mucosa from the same patient were used as controls. Polyclonal anti-human PA2.26 antibody was obtained by rabbit immunization with a synthetic peptide composed of amino acids 37 to 51 (P37–51) of the PA2.26 protein ectodomain. The method for antibody production has been fully described elsewhere.9 Anti-PA2.26 antiserum has been optimized and its expression analyzed in a wide variety of human tissues, with proven specificity for lymphatic endothelium. As the epitope recognized by this antiserum is not masked by formalin fixation, antigen retrieval methods are not needed. Briefly, 3-µm-thick paraffin-embedded tissue sections were deparaffinized in xylene, rehydrated through graded alcohols, washed with phosphate-buffered saline (PBS) and incubated at room temperature with anti-PA2.26 antiserum at 1:4000 dilution. The EnVision + TM-Peroxidase complex (HRP; DAKO A/S, Glostrup, Denmark) was used as the secondary antibody. The reaction was developed with diaminobenzidine substrate-chromogen solution (DAKO Co). Cell nuclei were stained for 15 s with Harris haematoxylin; the sections were dehydrated through a series of graded ethyl alcohols from 70 to 100% and were mounted in a permanent medium (Eukitt, O. Kindler GMBH & Co.; Freiburg, Germany).

Intratumoral lymphangiogenesis evaluation
Lymphangiogenesis evaluation was based on qualitative criteria, including the presence of initial lymphatic vessels within the tumour mass, their close association with tumor cells (Figs. 1A, BGo) and, finally, the presence of intravascular proliferation of the lymphatic endothelium (Figs. 1C, DGo).


Figure 1
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  		FIG. 1. (A) Numerous intratumoral initial lymphatic vessels (PA2.26 expression). Original magnification (OM) 4X. (B) ILV expressing PA2.26 antigen close to tumoral nests (OM: 40X). (C, D) The new lymphatic vessels show endothelial proliferation (OM: 40X). (E) Strong VEGF-C cytoplasmic expression in tumour cells (OM: 40x). (F) Negative VEGF-C expression in tumour cells; regenerative muscle cells are positive and were used as an internal control (OM: 10X).

 
Cases in which ILV was ascertained were divided into two groups according to the number of initial ILVs observed. Thus, high-grade lymphangiogenesis was attributed to samples with more than two initial ILVs in three fields at 20x magnification, and low-grade lymphangiogenesis was ascribed if there were two or fewer.

Evaluation of VEGF-C expression
VEGF-C assessment was performed in 75 of the 76 cases. Muscle cells in a regenerative state, peripheral nerve Schwann cells or macrophages were used as control tissues. VEGF-C immunostaining was carried out by the EnVision + TM-peroxidase method with antigen retrieval. Briefly, after deparaffinization and rehydration, sections were washed with PBS. Before endogenous peroxidase blocking, the antigen was retrieved by heating the slides in citrate buffer at pH 8 for 2 min in a pressure cooker. Slides were then incubated overnight with rabbit anti-VEGF-C antiserum (Zymed Laboratories; San Francisco, CA, USA) at 1:100 dilution; the complex EnVision + TM-peroxidase anti-rabbit (DAKO A/S, Glostrup, Denmark) was used as the secondary antibody, and the sections were developed and counterstained as described above.

Samples were evaluated by visual examination and assigned a subjective semiquantitative graded score for VEGF-C immunostaining intensity of cytoplasmic and granular staining that ranged from 0 to 3 (0: negative, 1: doubtful or very weak, 2: moderate and 3: strong).

As areas with different immunostaining intensity within the same section were often observed, the highest score was assigned as the overall immunohistochemical intensity. For ease of analysis, VEGF-C expression was differentiated as positive (2 and 3) or negative (0 and 1) (Figs. 1E, FGo).

Visual assessment of VEGF-C and PA2.26 antigen expression was performed separately by two pathologists who were unaware of the clinical characteristics of the studied patients. Conflicting results were resolved by consensus; in cases considered as doubtful, all evaluations were performed on new sections.

Statistical analysis
Summary statistics of individual variables were calculated: means and standard deviations for quantitative variables and percentages for qualitative ones. The association between quantitative and qualitative variables was studied by the Pearson {chi}2 or Fisher’s exact tests as appropriate. Student’s t test or analysis of variance (ANOVA) were used to compare the means of quantitative variables of two or more groups, respectively. Overall and disease-free survival times were estimated by the Kaplan-Meier method and the different subgroups were compared using the log-rank test. Construction of a number of Cox regression models controlled for confounding factors and interactions among time-dependent variables of this type. Unconditioned logistic regression was used for the multivariate analysis of relapse incidence. Statistical significance was assumed for values of P < 0.05. All statistical analyses were performed using SPSS version 10.0 (SPSS Inc., Chicago, IL, USA), in the Methodological Support Unit of the Foundation for Biomedical Research (Hospital Universitario de la Princesa, UAM).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Intratumoral lymphangiogenesis
Morphological signs of intratumoral lymphangiogenesis (IL) were apparent in 35/76 patients (46%); high-grade and low-grade lymphangiogenesis were observed in 16 and 19 cases, respectively.

Univariate analysis revealed a statistically significant association between the size of the primary tumour and the presence of IL: the larger the primary tumour, the greater the probability of occurrence of IL (P = 0.016). Lymphangiogenesis presence and grade were also linked to relapse: IL was found in 9/10 patients (90%) with lymph node relapse; in eight of whom, the IL was low-grade. IL was not associated with primary tumour location, histological grade or age (Table 2Go).


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  		TABLE 2. Univariate analysis of the relationship between intratumoral lymphangiogenesis and clinicopathological variables
 
VEGF-C expression
VEGF-C expression was positive in 45/75 patients (60%). This factor was significantly associated with relapse: 90% of patients with relapse had VEGF-C-positive expression (P = 0.044). This character was unrelated to the presence or grade of IL (P = 0.143), although there tended to be more lymphatic vessels in VEGF-C-positive tumours, and 80% of cases with high-grade IL expressed VEGF-C (Table 3Go). No association was found with tumour histological grade or location, or age.


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  		TABLE 3. Univariate analysis of the relationship between VEGF-C expression and clinicopathological variables
 
Survival analysis
None of the studied variables had a statistically significant association with overall survival. However, our model revealed that the presence and grade of IL (P < 0.001), and VEGF-C expression (P = 0.038) were associated with a shorter disease-free period. This relationship was confirmed by the multivariate analysis, in which patients with low-grade IL and positive VEGF-C expression showed, respectively, 16.2-fold and 8.44-fold shorter disease-free survival (DFS) periods (P = 0.009 and 0.045; Table 4Go, Figs. 2A, BGo).


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  		TABLE 4. Univariate and multivariate analyses of disease-free survival in relation to clinicopathological variables
 

Figure 2
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  		FIG. 2. Low-grade intratumoral lymphangiogenesis (A) and VEGF-C expression (B) are associated with DFS shortening.

 
Lymph node relapse analysis
Relapses occurred in 10 patients (13.1%), all occurring in regional cervical lymph nodes. Patients 1, 5, 6, and 9 died, but only in patients 6 and 9 were the deaths tumour related. The characteristics of the relapses are described in Table 5Go. IL and VEGF-C expression were significantly associated with the occurrence of cervical lymph node relapse (P < 0.001 and P = 0.044, respectively). These results were confirmed by multivariate logistic regression, which revealed that low-grade IL and positive VEGF-C expression respectively increased the probability of relapsing in cervical lymph nodes by 30.3- and 16.24-fold (P = 0.005 and P = 0.032). A trend towards cervical relapse (P = 0.087) was also observed in patients with the largest tumours (Table 6Go).


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  		TABLE 5. Descriptive summary of relapses
 

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  		TABLE 6. Univariate and multivariate analyses of the relationship between relapse and clinicopathological characteristics
 
In our study univariate and multivariate analyses failed to show any association between post-surgical treatment (radiotherapy) and disease-free survival (P = 0.685) or lymph node relapse (P = 0.638) (Tables 4Go and 6Go).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The term lymphangiogenesis refers to the development and proliferation of new lymphatic vessels emerging from the host vasculature. This event takes place in the embryo during processes of tissue repair and inflammation, and in parasitic infections and tumours.7

The study of lymphatic vessels is difficult because they cannot be reliably distinguished from blood vessels. The discovery of markers specific to lymphatic endothelium in the last few years has markedly improved our knowledge of lymphatic microcirculation.10 These new markers include VEGFR-3,11–14 podoplanin,10,15 Prox-116 and LYVE-1.2,17,18

The antigen PA2.26 is a newly established lymphatic endothelial marker. It was identified in our laboratory as a small mucin-like transmembranal glycoprotein of 172 amino acids in length. Anti-PA2.26 antiserum shows high affinity for an epitope on the extracellular moiety that is not masked by formalin fixation. It is very useful for the identification of paraffin-embedded lymphatic vessels and has been used in a previous investigation.19 Characterization of mouse and human PA2.26 antigen cDNA sequences9,20 revealed that PA2.26 is identical to podoplanin, a glycoprotein expressed in alveolar type I cells and glomerular podocytes,21,22 which is also a specific marker for lymphatic endothelium.15

Role of VEGF-C in tumours
To date, two lymphangiogenic factors, VEGF-C and VEGF-D, have been identified within the VEGF family; their receptor is the tyrosine kinase-type receptor, VEGFR-3.23,24

Evidence of VEGF-C expression in various human tumours has been reported,25 including head and neck squamous cell carcinomas from several locations such as the larynx and hypopharynx.26–30 Most studies have established a statistically significant relationship between VEGF-C levels in primary tumour and regional lymph node metastases.23,30,31

The results obtained from a number of experimental studies32–37 have unequivocally shown that VEGF-C induces lymphangiogenesis in the primary tumour, which, in turn, promotes metastasis development in lymph nodes. Some researchers believe that VEGF-C may play additional roles to lymphangiogenesis. It is possible that VEGF-C favours lymphatic endothelium activation, thereby inducing the secretion of chemokines and/or similar factors that are able to attract tumour cells, and facilitating their entrance into the initial lymphatic vessels.25 VEGF-C may also be involved in angiogenesis,38 the increase of vascular permeability and macrophage chemotaxis.24 The first two may favour metastatic dissemination by means of an increase in interstitial tumour pressure that would force the cells to enter the lymphatic vessels. Macrophage chemotaxis and the attraction of macrophages towards the tumour periphery might promote the metastatic process through the supplementary VEGF-C excreted by the macrophages. The various biological effects of VEGF-C on tumours may depend on its processing by the tumour cells.38 Although recent investigations performed in patients with colorectal cancer39 and oral carcinoma40 suggest that VEGF-C expression might be able to induce lymphangiogenesis in human tumours and lead to an increased number of lymph nodes metastases, this hypothesis is not yet proven.7,24,41 The significance and role of VEGF-C in the process of lymph node metastasis development in human tumours should be established by future studies. Our results are consistent with those observed by Beasley et al.26 in that we also found no statistical association between VEGF-C expression by tumour cells and the incidence of IL, although we did observe a trend towards the presence of more initial intratumoral lymphatic vessels (ILVs) when VEGF-C was expressed. We also observed a significantly higher incidence of lymph node relapses when VEGF-C was expressed, which is consistent with the findings of O-Charoenrat et al.27 and Neuchrist et al.28

Tumour lymphangiogenesis
Until recently, lymphangiogenesis was thought to be a missing event in cancer, and most tumours were believed to lack lymphatic vessels.25 The identification of markers specific to lymphatic endothelium and their growth factors has enabled these concepts to be reconsidered. To date, IL has been observed solely in experimental tumours23,25 and a few types of human tumours, particularly primary melanoma,42 head and neck at different subsites19,26 and, as shown in the present study, laryngopharyngeal carcinoma. Further research is needed to determine whether IL is restricted to specific tumour types, and to elucidate whether its presence has prognostic significance. In this sense, the presence of IL in early oral carcinoma has been found to be associated with disease relapses,19 while a high density of ILV has been linked to cervical lymph node metastasis in oropharyngeal carcinoma, but not in oral cavity or laryngeal cancers.26 In our study, low-grade IL was significantly associated with the occurrence of lymph node relapses, which is consistent with results previously reported for melanoma.42

The role of ILVs in tumour dissemination is under debate.35,43 Several studies have reported a link between the number of tumour-associated lymphatic vessels and the presence of lymph node metastasis.25,26 In the present study, low-grade IL was significantly associated with cervical lymph node relapse, in contrast with cases with negative or, surprisingly, high-grade IL. The increase in the lymphatic vessel density has been associated with good prognoses in cutaneous melanoma and cervical carcinoma.42,44 These results could be explained by an enhanced immune response to the tumour cells. Supporting this hypothesis, cases of high lymphatic vessel density have been reported to be frequently associated with an augmented lymphocytic infiltrate. We have also observed that high-grade lymphangiogenesis is frequently linked with an intense inflammatory infiltrate. We believe that this infiltrate may act as a supplementary source of VEGF-C, leading to an increase in vascular permeability and tumour interstitial pressure that would randomly induce some tumour cells to enter the initial lymph vessels passively. In contrast, active recruitment of tumour cells by the initial lymph vessels could predominate in the cases of low-grade lymphangiogenesis. This hypothesis needs to be investigated further.

The metastatic process clearly consists of a complex series of steps and interactions between the primary tumour and the patient’s characteristics, among which lymphangiogenesis and VEGF-C secretion are probably necessary but not sufficient conditions.31 The present study reveals the important influence of these factors on cervical lymph node relapse. Our results show that low-grade IL and VEGF-C expression by tumour cells are independent predictors of the risk of cervical lymph node relapse in patients with laryngopharyngeal squamous cell carcinoma (T1–T4) N0. Further studies aimed at confirming these results and evaluating their diagnostic application to the identification of patients at higher risk of lymph node metastases are warranted. This would enable us to select more accurately the most suitable therapeutic strategy for healing the disease while avoiding overtreatment.


   ACKNOWLEDGMENTS
 
We thank Dr. Rodríguez-Salvanés for reviewing the statistical analyses and Dr. Phil Mason for his invaluable help with the English language. This work was supported by grants from the Thematic Network for Cooperative Research RESPIRA (Code C003/011; Foundation for Biomedical Research, Hospital Universitario de la Princesa) and the Fondo de Investigaciones Sanitarias (Code FIS 02/1025).

Received for publication July 13, 2006. Accepted for publication July 20, 2006.


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TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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