ARTÍCULO CIENTÍFICO

 

Early stage oral cancer: prognosis with regard to histological grading, intratumoral lymphangiogenesis, and the expression of vascular endothelial growth factor-C (VEGF-C)

Estadios precoces de cancer oral: pronóstico en relación con gradación histológica, linfagiogénesis intratumoral y expresión de factor de crecimiento endotelial vascular Tipo-C (VEGF-C)*

 

 

M.F. Muñoz-Guerra¹, A.L. Capote Moreno¹, E.M. Gómez Marazuela, C. Gamallo Amat³

1 Médico Adjunto. Servicio de Cirugía Oral y Maxilofacial (Jefe de Servicio- F.J. Díaz González), 
Hospital de la Princesa, Universidad Autónoma de Madrid (U.A.M.), Madrid.
2 Departamento de Bioquímica, Facultad de Químicas, Universidad Complutense, Madrid.
3 Profesor. Departamento de Anatomía Patológica, Facultad de Medicina, Universidad Autónoma de Madrid (U.A.M.), Madrid.

* Premio "Gómez Iglesias" 2004

Dirección para correspondencia

 

 

 

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ABSTRACT

Objectives. Histological grading systems have been classically used as a prognostic factor and clinical behavior markers in oral squamous cell carcinoma (OSCC). However, their prognostic usefulness remains controversial. Our aim was to evaluate the presence of intratumoral lymphangiogenesis (IL), a new morphological finding, in a retrospective analysis of paraffin embedded tissue samples that corresponded to a group of early stage oral squamous cell carcinoma cases, and to relate this with histological grading systems while keeping in mind their prognostic significance. We also wanted to determine if the expression of vascular endothelial growth factor-C (VEGF-C) is correlated with the evolution of the disease.
Design. We performed a retrospective analysis of 96 patients with OSCC. All cases were T1-T2 neoplasms and were treated primarily by local resection and elective neck dissection that showed no neck involvement. In the group of 96 specimens, we analyzed IL using the specific marker PA2.26 for lymphatic endothelium. Also, we studied the expression of (VEGF-C). All cases were classified according to the histological grading systems described by Broders, Anneroth and Bryne. The statistical analysis was based on the univariate analysis of cause-specific survival and disease recurrence free-survival according to the Kaplan-Meier method.
Results. The group of patients without intratumoral lymphangiogenesis showed a better prognosis with regard to survival and disease- free period, but the difference was not statistically significant. The study showed no relationship between VEGF-C expression and the presence of intratumoral lymphangiogenesis. However, no recurrences were observed in the group without VEGF-C expression. The comparative analysis of the histological grading system showed a statistical relationship between the Broders and Anneroth systems (p<0.01) and between the Broders and Bryne systems (p<0.001). Our study demonstrated an inverse relationship between the values of the Anneroth system (p<0.01) - Bryne system (p<0.001) and the presence of intratumoral lymphangiogenesis.
Conclusion. The clinical value of the histological grading systems can increase by including new parameters that take into account the biological behavior of the tumor. Expression of VEGF-C and intratumoral lymphatics may thus be useful prognostic markers for oral squamous cell carcinoma.

Key words: Oral cancer; Prognostic factors; Grading systems; Lymphangiogenesis; Vascular endothelial growth factor.

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RESUMEN

Objetivos. Los sistemas de gradación histológica se han usado clásicamente como factor pronóstico y marcadores de comportamiento clínico en el carcinoma epidermoide intra-oral (CEI). Sin embargo, su utilidad pronóstica permanece controvertida. Nuestro objetivo ha sido evaluar la presencia de linfangiogénesis intratumoral (LI), un nuevo hallazgo morfológico, en un análisis retrospectivo de muestras de tejido en parafina dentro de un grupo de estadios precoces de CEI, relacionándolo con clásicos sistemas de gradación histológica y teniendo en cuenta su importancia pronóstica. Asimismo, pretendemos determinar si la expresión del factor de crecimiento endotelial vascular –C (VEGF-C) se correlaciona con la evolución de la enfermedad.
Diseño. Realizamos un estudio retrospectivo basado en 96 casos de CEI. Todos los pacientes presentaban tumores intraorales T1-T2 y fueron tratados primariamente mediante resección local asociada con disección cervical electiva, la cual mostró ausencia de afectación ganglionar regional. En el grupo de 96 muestras analizamos la LI utilizando el marcador específico del endotelio linfático PA2.26. Adicionalmente, estudiamos la expresión del VEGF-C. Todos los casos fueron clasificados de acuerdo con los sistemas de gradación histológica descritos por Broders, Anneroth y Bryne. El estudio estadístico se fundamentó en el análisis univariante de supervivencia causa-específica y supervivencia libre de recidiva según el método de Kaplan-Meier.
Resultados. El grupo de pacientes con ausencia de LI mostró mejor pronóstico en cuanto a supervivencia y periodo libre de enfermedad, aunque la diferencia no mostró valores estadísticamente significativos. El estudio no mostró una relación entre la expresión de VEGF-C y la presencia de LI. Sin embargo, no observamos recidivas entre el grupo con ausencia de expresión de VEGF-C. El análisis comparativo de los sistemas de gradación histológica mostró una relación estadísticamente significativa entre los sistemas de Broders y Anneroth (p<0,01) y entre los sistemas de Broders y Bryne (p<0,001). Nuestro estudio demostró una relación inversa entre los valores de los sistemas de gradación Anneroth (p<0,01) – Bryne (p<0,001) y la presencia de LI.
Conclusión. El valor clínico de los sistemas de gradación histológica puede incrementarse si se incluyen nuevos parámetros que consideren el comportamiento biológico del tumor. La expresión de VEGF-C y la presencia de linfáticos intratumorales pueden ser marcadores marcadores pronósticos de utilidad en el CEI.

Palabras clave: Cancer oral; Factores pronósticos; Sistemas de gradación; Linfangiogénesis; Factor de crecimiento endotelial vascular.

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Introduction

The prognostic determination of oral squamous cell carcinoma (OSSC) is usually based on clinical1 or pathological^2-4 classifications, although most professionals currently use the TNM clinical classification system as a prognostic marker. Different authors have devised various histological grading systems with a view to making more precise estimates as to the biologic behavior of certain neoplasms.^{2, 5, 6} However, there is currently controversy as to the real use of these grading systems.⁷

In patients with small SCC (T1-T2) a good prognosis can be expected with a survival rate of 65-95% of cases.^{8, 9} However, it should be taken into account that the percentage of patients with a bad prognosis can reach 35% of cases. In small tumors, the investigation of new factors with predictive capacities to facilitate a possible prognosis is therefore necessary. These would express cell aggressiveness independently of tumor volume.^{10, 11}

The presence or absence of lymph node involvement at diagnosis is a basic prediction factor as to the course of head and neck tumors, and it affects the individual treatment of each patient decisively. Understanding the underlying mechanisms of SSC and its capacity for metastasizing via the lymphatics has lately turned into an attractive field for investigation^12-14 Our previous studies¹⁵ correlate the presence of lymphatics within tumor tissue with poor SCC prognosis. Moreover, a recent study by Kyzas and cols.¹⁶ correlated the presence of neoplastic cell emboli in the interior of intratutemas moral lymphatics with a high rate of tumor related mortality.

The molecular mechanisms that stimulate lymphangiogenesis are not completely clear although certain studies have suggested that the expression of vascular endothelial growth factor C (VEGF-C) within the tumor tissue can stimulate endothelial cells to generate new lymphatic vessels.¹⁷ The purpose of this work was to analyze a group of patients with early stages of SCC and to find a possible correlation between intratumoral lymphangiogenesis (IL), the expression of VEGF-C and the prognosis of the disease. We therefore used the three traditional histological grading systems (Broders, Anneroth and Bryne) correlating the values obtained with the presence of IL and with the prognosis of the disease.

 

Material and methods

Patients

The Department of Oral and Maxillofacial Surgery of our hospital has a database including 500 patients that were surgically operated of oral squamous cell carcinoma over the period 1985-2004. Of the global group appearing in this database, a subgroup of 96 cases was obtained with early stage tumors (I-II) that had a follow-up of more than 3 years. All the cases in this subgroup were treated primarily by means of local surgery with elective neck dissection. The pathological analysis of the specimens from the neck dissection did not show any metastatic nodal involvement in this group of patients (pN0). The information in the database that was used for this study included age, sex, primary location of the tumor, disease stage, pathological findings, presence of relapses (including the location), disease-free interval and global survival. Patient follow-up was carried out monthly over the first year, every three months during the second year and every six months as from the third year.

Methods

The surgical specimens from the patients in our study archived in the Department of Pathology in our hospital, were analyzed. All the samples were fixed in 10% formalin and embedded in paraffin. For study purposes, the most representative samples of the tumor were used, as well as normal tissue as a control.

Intratumoral lymphangiogenesis (IL)

Immunohistochemical staining
The polyclonal antibody against human PA2.26 was obtained by means of immunization in a rabbit with a synthetic peptide comprising amino acids 37-51 (P_37-51) of the protein ectodomain PA2.26. The method of antibody production has been described in detail previously.^15,18 The anti- PA2.26 antiserum has been optimized, and its expression analyzed in large amounts of human tissue, and the specificity for lymphatic endothelium has been demonstrated The epitope that recognizes this antiserum is not masked by formalin and, for that reason, heating techniques are not required. This facilitates the normalization of the technique and, therefore, the reproduction of the results. Once the sections of tissue with a thickness of 5 µm. had been deparaffinized, the Envision+-peroxidase method (Dako A/S; Glostrup, Denmark) was used for the immunohistochemical study. Incubation was carried out during an hour with the anti- PA2.26 antiserum with a 1:400 dilution at room temperature before and after preincubation with P_37-51 and P control as a negative control. The sections were then dehydrated in graded alcohols, cleared with xylol and mounted in Permount (Fischer Sc.; Pittsburg, U.S.A.) after counterstaining with hematoxylin. Using this marker we were able to carry out a descriptive analysis of the lymphatic capillaries, and the IL cases were identified.

IL evaluation
In the descriptive analysis of the microcirculation architecture, the presence of peritumoral or intratumoral lymphatic capillaries was assessed, although by the study only cases with lymphatics inside the tumor tissue were considered. Tissue specimens obtained from healthy oral mucosa were used as control specimen.

Cases were included in the intratumoral lymphangiogenesis group IL(+) if lymphatic vessels were observed within the tumor tissue, in intimate contact with the tumor cells (Fig. 1A). Cases were considered to be in the IL(-) group if lymphatics were not observed on the inside of the tumoral tissue (Fig. 1B).

Vascular endothelial growth factor-C (VEGF-C)

Immunohistochemical technique
Immuno-staining with VEGF-C was carried out using the Envision+-peroxidase method (HRP, Dako A/S, Glostrup, Denmark) with antigen retrieval by heat and citrate buffer pH8. Briefly, the paraffin-embedded samples were initially deparaffinized with xylol. They were re-hydrated through descending concentrations of alcohol and later washed in PBS (phosphate buffer saline). Next, endogenous peroxidase was blocked and antigen retrieval was carried out using a pressure cooker for two minutes. The process was then continued with overnight incubation with rabbit anti-VEGF-C serum (Zymed Lab.; San Francisco, California, U.S.A.) using a 1/100 dilution. The anti-rabbit Envision+- peroxidase complex was used as a secondary antibody. The antigen-antibody reaction produced was detected using amino-benzidine and H₂O₂. The nuclear cells were stained with Harris’ hematoxylin stain (15 seconds) and the samples were dehydrated in increasing concentrations of alcohol and mounted in a permanent medium (Eukitt)(O. Kindler GMBH&Co; Freiburg, Germany).

VEGF-C evaluation
VEGF-C immunoreactivity was examined by two of the authors (A.L.C. and C.G.A.) who were not aware of the clinical results of each case. Only 62 cases out of the global group could be evaluated. In order to evaluate the intensity of the VEGF-C stain in the SSC, a tumor free region of the epithelium was used as internal control. VEGF-C expression in neoplastic tissue was defined as negative if positive cells made up 0-25% of the tissue, and positive if positive cells made up >25%. Thus, the expression was considered as present or absent without taking into account immunostaining intensity.

According to the morphology of the expression factor, another two variables were obtained:

• Expression distribution (Fig. 2): This was considered homogeneous or heterogeneous depending on if the morphology of the expression was uniform or not.

• Expression location (Fig. 3): This was considered as central, peripheral or diffuse depending on the predominant location of the expression in the tumor nests.

Histological grading systems In all cases, the main section that included the full thickness of the tumor tissue (with the invasion margins) was used. The histological grading was analyzed using the following classification systems:

• Broders’ classification:⁵ this system is based on the degree of differentiation and keratinization of tumor cells. According to this, tumors were considered well, moderately or poorly differentiated.

• Anneroth classification:² This is a point system that takes into account the following parameters: degree of keratinization, nuclear polymorphism, number of mitoses per high powered field, pattern and stage of invasion as well as the degree of lymphoplasmacytic infiltration (Table 1).

• Bryne’s classification:⁶ an identical system to that of Anneroth with the exception that the «number of mitoses» is omitted.

Statistical analysis of the results

With the aim of facilitating statistical analysis of the results, and taking into account the original score according to the Anneroth and Bryne systems, we analyzed the following dichotomic variables:

• Anneroth-Transformed (Annertrans).

- Value I- Anneroth <=13
- Value II-Anneroth >13

• Transformed Bryne (Brynetrans).

- Value I- Bryne <=9.
- Value II- Bryne >9.

Next, as a result of the high degree of subjectivity of some the variables in these grading systems, and in order to facilitate the statistical analysis, we considered appropriate transforming into dichotomies four of the variables analyzed, as follows:

• Transformed-Keratinization (Keratrans).

- Value I- High or moderate keratinization.
- Value II-Poor or no keratinization.

• Transformed Polymorphism- (Politrans).

- Value I- Scarce or moderate polymorphism.
- Valor II-Abundant or extreme polymorphism.

• Transformed Mitoses rate (Mitosestrans).

- Value I- Mitoses rate 0-3.
- Value II- Mitoses rate >3.

• Transformed lymphoplasmacytic infiltration (Infiltrans).

- Value I- Marked or moderate lymphoplasmacytic infiltration.
- Value II-Slight or no lymphoplasmacytic infiltration.

 

Statistical method
The correlation between the different clinical and pathological parameters was studied by means of a χ₂ test with Yates’ correction. Cause-specific survival was considered as the period between the surgical intervention and the death of the patient through tumor disease. Relapse-free survival was used for measuring prognosis and this was considered as the period between the surgery and the relapse date, or the date of the last followup. The univariate analysis of cause-specific survival and relapse-free survival was based on the Kaplan-Meier method, using the log-rank test for comparative study. The p<0.05 values were considered as statistically significant. For the statistical study the SPSS 8.0 program (SPSS Inc., Chicago, IL, U.S.A.) was used in all cases.

 

Results

The global group (96 cases) included 49 pT1 (51%) and 47 pT2 (49%). The mean age observed was 57.25 ± 14.06 years. The study included 69 males (71.9%) and 27 females (28.1%). In the follow-up, there were 16 cases (16.7%) of relapse: 6 cases at a local level (6.3%) 8 neck cases (8.3%) and 2 local neck cases (2%).

Intratumoral lymphangiogenesis
The analysis of the samples with PA2.26 immuno-staining was found to be a constant marker for lymphatic endothelium. The findings as to peritumoral lymphangiogenesis were practically constant, while in the group with 96 cases, 57 cases (59.4%) had typical IL. The analysis of this group showed a greater rate of relapse in the IL(+) group, although the difference could not be considered as statistically significant (Table 2). The univariate analysis by means of the log-rank test showed a better disease-free interval for the IL(-) group (Fig. 10). In the global group, 6 deaths occurred during the follow-up, 5 cases of the IL(+) group and one case of the IL(-). The gender analysis showed a greater rate of IL(+) in the female sex with a difference that was statistically significant (p<0.01) (Table 3). It should be pointed out that only 1 death was observed in the female group, which was a case of the IL(+) group (1/22 cases-4.5%). Of the male patient group with IL(+) we observed 4 deaths (4/35- 11.4%).

 

With regard to VEGF-C expression, 51 cases showed positive expression while the VEGF-C(-) was made up of 11 cases. Within the 62 cases analyzed, 11 showed relapse during the follow-up period, and they all were included in the VEGF-C(+) group. The analysis of the disease-free interval showed better results for the VEGFC group, although the difference did not result in statistically significant values. The differences in relation to the site and the distribution of this vascular growth factor expression were not statistically significant with regard to the existence of relapses during the followup period. VEGF-C expression did bear a significant relationship with the presence of intratumoral lymphatics (Table 4). Within the IL(+) VEGF-C(+) subgroup, 15 cases (46.9%) showed a diffuse expression of this factor, 12 (37.5%) had central expression and 5 (15.6%) showed peripheral expression. With regard to distribution, 19 cases (59.4) were considered homogeneous and 13 cases (40.6%) were heterogeneous.

 

Correlation of histological grading systems with intratumoral lymphangiogenesis
With regard to Broders’ classification, of the 96 cases analyzed, 38 were considered well-differentiated, 52 moderately differentiated and 6 poorly differentiated. A statistically significant correlation was not observed between lymphangiogenesis and Broders’ histological grading. With regard to relapse, there were 8 occurrences in patients with welldifferentiated tumors (8/38- 21% of cases) and another 8 with moderately differentiated tumors (8/52- 15.4% of cases). None of the patients with poorly differentiated tumors suffered a relapse during the follow-up period.

The comparative study of the different histological grading systems showed a statistically significant relationship between the Broders’ and Anneroth systems (p<0.01), and between the Broders’ and Bryne systems (p<0.001) (Table 5). On correlating the Anneroth grading system with the occurrence of relapse at follow-up, 8 relapse cases were observed in the Annertrans value II group (8/44- 18.1%) (4 local, 3 neck and 1 local neck case). In the Annertra value I group 8 relapses were also observed (8/52- 15.3%) (2 local, 5 neck and 1 local neck case). Eight relapses were also observed in the Brynetrans value I group as well as in the Brynetrans value II group. With regard to the survival curve during the disease-free interval, no log-rank values were observed that were of significance for either the ANNERTRANS variable (p=0.73), nor for the Brynetrans variable (p=0.91). With regard to survival, 3 deaths were observed in the value I group as well as in the value II of theAnnertrans and Brynetrans variables.

The statistical analysis did not establish a link between the Keratrans (p=0.85), Polytrans (p=1), Mitosetrans (p=0.63), Infiltrans (p=0.7) variables and the presence of relapse at follow-up. Also, no relation was established between the invasion mode (p=0.57) or degree of invasion (p=0.85) and the presence of recurrence.

The analysis that corresponded to the relationship between the different variables of the Anneroth classification and IL resulted in the following data:

• An inverse relationship was appreciated between the Keratrans variable and the presence of IL (p<0.01) (Table 6).

• There was no relationship between Polytrans (p=0.36) and Mitosestrans (p=0.8), mode of invasion (p=0.25) variables and IL.

• The degree of invasion and the IL variables did not show a relationship that was statistically significant, although the cases of muscular invasion showed a high percentage of IL (34/52-65.4% of cases).

• A relationship that was statistically significant was observed between the presence of IL and the Infiltrans variable (p<0.01) (Table 7).

• A relationship that was statistically significant was appreciated between the lower values of Annerttrans (p<0.01) – Brynetrans (p<0.001) and the presence of IL (Tables 8 and 9).

 

 

Discussion

A significant percentage of patients with early stages of SCC have a poor prognosis despite the small size of the tumor. In fact, Bundgaard and cols.¹⁹ demonstrated that up to 25% of patients with T1 could show poor prognosis at follow-up. The current standard treatment for oral tumors when the probability of neck involvement is greater than 20%, is local resection and even elective neck dissection for clinically negative necks. However, there is great controversy as to the indication of elective dissection for stage I-II oral cancer, which shows the importance of finding prognostic factors for early stage SCC in particular. In a study carried out by Beenken and cols.²⁰ based on 169 patients with early stage lingual carcinoma, the authors found a rate of local or cervical relapse of more than 20% regarding the cases treated primarily with local resection only. The percentage of false negatives, that is to say, N0 and pN+ necks, varies between 15 and 60% depending on the series.^{21, 22}

The objective of the tumor grading systems is to classify patients into groups so that a similar clinical course can be expected. The current TNM classification is the only widely used system for predicting the clinical result of SCC. The TNM system includes acceptable prognostic parameters but the biological properties of the tumor cannot be predicted. With the aim of evaluating these biological properties, histological grading systems emerged.

In 1920, Broders⁵ devised a histological classification system that he applied to squamous cell carcinoma of the lip. Four categories were identified depending on the proportion of neoplasm resembling the normal epithelium. The classification system was later modified by a group of pathologists who identified only 3 tumor types. The system developed by Broders divided tumors into three types according to the degree of differentiation: well-differentiated, moderately differentiated and poorly differentiated. He showed how the rate of metastasis increases in the groups of squamous cell carcinoma of the lip from well-differentiated tumors to poorly differentiated tumors. This system has been widely used, but its predictive value with regard to metastasis or survival has proved to be limited.²³ The results of our study also show the limited value of this classification system. The lack of correlation between Broders’ grading system and prognosis can be explained by the fact that SCC is usually made up of a heterogeneous cell population with different degrees of invasiveness depending on the tumor area analyzed.

The limited use of Broders’ system led different authors to develop new systems for multifactorial classification.^{24, 25} In 1966, Arthur and Fenner²⁶ observed a significant correlation between the degree of keratinization, mitotic activity, hyperchromatism and the prognosis for tongue cancer, and they suggested that the evaluation of different parameters within a malignancy classification system could be useful. In 1973, Jakobsson and cols published a multifactorial grading system in order to evaluate tumor tissue morphologically. ²⁴ Anneroth and cols. modified this system by evaluating morphological criteria more specifically.²⁵ In 1991 Bryne and cols.²⁷ introduced a multifactorial grading system that took into account tumor invasion margins while the author eliminated the «mitosis count» factor because of the controversy surrounding the use of this factor as a result of cellular heterogeneity and inter-observer subjectivity.

Al-Rajhi and cols.²⁸ in a series of 85 patients with tumors of the mobile tongue appreciated that neither the degree of differentiation nor tumor size appeared to affect loco-regional control or survival. Umeda and cols.²⁹ studied a group of 60 cases with ISCC and surprising results were observed: the presence of neck metastasis was not correlated with either primary site nor with tumor size, but a correlation was found between histological grade of malignancy (WHO grading) and the prevalence of neck metastasis. Okamoto and cols.³⁰ studied the development of delayed neck metastasis in a group of 59 T1-T2 patients that were treated by means of local surgery only. These authors did not find a statistical correlation between the histological grade of malignancy (as defined in Broders’ parameters) and the incidence of relapse; however, VEGF-C expression did influence the development of neck node metastasis in the follow-up period. Lim and cols.³¹ observed contradictory results as their series of 56 stage I-II patients, their study showed a statistical correlation between Broders, Anneroth and Bryne with late cervical metastasis. Our series particularly focuses on tumors of less than 4 cm and with no cervical node involvement. In this homogeneous group of patients, our results showed the little value of the different grading systems with regard to disease-free intervals and survival.

Our study did not reveal any statistical correlation between the different parameters used for evaluating histological grading and the course of the disease. However, of all these, mode of invasion had the greatest prognostic importance. Crissman and cols.³ demonstrated in a study based on 77 patients that were treated by means of preoperative radiation therapy and surgery, that the mode of invasion was the only histological factor that could predict survival. The neoplasms that invaded as solid cords had a better prognosis than when invasion was in the form of thin cords. In a study published in 2000 on 122 cases of different tumor sizes, López Pizarro³² showed in a logistic regression analysis how, of the factors analyzed, the pattern of invasion had the greatest metastatic risk.

The prognosis in oral cancer has been related for more than 20 years with different scoring systems with the intention of being predictive. In 1987, Gavilán and colls.³³ published a study on more than 60 factors that could affect the prognosis of carcinomas of the head and neck; in spite of the fact that the study only made use of 11 of the factors analyzed, the study was not useful clinically. Other scoring systems have tried to relate various factors depending on the primary tumor with the risk of neck node involvement. The system that is used most was created by Martinez-Gimeno after analyzing a group of 126 patients with squamous cell carcinoma of the oral cavity and hypopharynx.³⁴ The risk of cervical lymph node metastases was correlated with 7 parameters: T stage, vascular invasion, tumor grading, tumor thickness, tumor-host interphase, inflammatory infiltration and perineural spread. However, despite Yuen and cols³⁵ pointing out that the predictive value of this system was based on the weight given to the «tumor thickness» parameter, the system has been validated by a recent work of its author.³⁶

Our study did not include a statistical analysis about the importance of tumor thickness in the prognosis of the disease. This trascendence has been demonstrated by previous. In fact, O´Brien and cols.³⁷ suggested in a recent work that elective neck dissection was necessary for tumors with more than a 4 mm thickness. Kurokawa and cols.³⁸ observed that tumors with a thickness >4 mm. and moderately differentiated, were associated with a high rate of cervical metastasis. In a study carried out by Yuen and cols.³⁹ that analyzed various factors traditionally considered as prognostic factors in patients with T1-T2 SSC, tumor thickness was the only predictive factor with regard to recurrence and survival. It should be pointed out the use of tumor thickness for prognosis can justify the need for adjuvant therapies but, with regard to indications as to the type of surgery to be carried out, it does not appear to be a useful parameter due to the fact that this measurement can only be obtained reliably if the specimen is completely resected. In our opinion, tumor thickness is not the only factor to be considered. The biological characteristics of the neoplasm should also be taken into account, as well as factors such as lymphangiogenesis or expression of tumor growth factors.

Previous studies, such as those carried out by Dantas and cols.⁴⁰ have showed no correlation between histological scores of malignancy and prognosis in oral carcinoma. The Anneroth classification system showed limited prognostic value in this study, as occurred in ours, but in contradiction to the results of other authors.^41,42 One of the problems of these classification systems was pointed out by Crissman and cols.³ who highlighted the limited value of variables that were difficult to interpret and exposed to subjectivity such as mitotic count and nuclear atypia. Previous studies by our investigation group have shown the advantages of using parameters less frequently employed such as the expression of epithelial adhesion molecules,⁴³ the presence of lymphatics in the tumor, and even the observation of lymphatic embolization by tumoral cells.¹⁵

Investigations that focus on understanding the biology behind lymph vessels have been enhanced by the discovery of specific lymphatic endothelium markers such as podoplanin, LYVE-1, prox-1 and VEGFR-3.^12-14 Among these markers we can include PA2.26, a cell-surface protein related to epidermal carcinogenesis and with the cutaneous remodelling process.^18,44 Previous reports have described a significant correlation between VEGF-C expression, lymphangiogenesis and regional metastases in certain types of tumors.^45-48 However, authors such as Beasley and cols.⁴⁹ have indicated that only certain types of neoplasms can use intratumoral lymphatics for developing metastasis. The difference in invasive potential could be due to the production of factors such as metalloproteinases⁵⁰ or vascular endothelial growth factors.⁵¹ The absence of intratumoral lymphatics in neoplasms that develop metastasis via the lymphatics, such as in the case of breast cancer⁵² can reflect the genuine differences between the behavior of different tumors, depending on histological type.

In this study, we analyze the expression of VEGF-C in surgical samples of OSCC cases, using immunohistochemical techniques and correlating these with the presence of intratumoral lymphatics and with the prognosis of the disease. Kishimoto and cols.,⁵³ in a study of 38 patients with early stage oral carcinoma, correlated the expression of VEGFC with the presence of regional lymph node metastasis. However, a data is specially significant, this correlation could not be established in patients with T3-T4 tumors. These results show that the expression of this endothelial growth factor can have different significance depending on the size of the tumor studied.

The combination of a simple addition method for the multifactorial grading systems should be viewed as not very logical. Without the analysis of the specific independent weight of each valuable the problem cannot be approached properly. According to our results, the data of multiple histological parameters can be associated and a combined score can be reached that can even turn out to have even less prognostic value than each of the factors on their own. Independently, the studies on biological factors and tumor growth in OSSC should be centered on groups of patients with small tumors, as the possibilities of independent treatment are greater. Those cases with unfavorable histological prognosis markers could be candidates for more aggressive treatment initially.

 

Conclusions

1. The presence of IL can be considered a risk factor with regard to the presence of relapses at a loco-cervical level in early stage SCC of the oral cavity.

2. In order to confirm a link between IL and the expression of VEGF-C more studies are necessary based on a larger number of cases, preferably with different tumor sizes.

3. The clinical usefulness of these prognostic systems in oral cancer increases if factors are included that take into account the biological behavior of the tumor.

 

Acknowledgements

The authors would like to thank Teresa Alcalá Zamora and Eva García for their technical assistance, as well as all the present and past members of the Department of Oral and Maxillofacial Surgery of our Hospital for their assistance in tabulating and compiling the data corresponding to the patients included in our study.

 

 

Dirección para correspondencia
Mario Fernando Muñoz Guerra
Servicio de Cirugía Oral y Maxilofacial
Hospital de la Princesa- Universidad Autónoma de Madrid, España
c/ Diego de León, 62
28006 – Madrid, España
e-mail: maxmferm@excite.com

Recibido: 22.08.2005
Aceptado: 24.02.2006

 

 

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