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Expression of vascular endothelial growth factor-C does not predict occult lymph-node metastasis in early oral squamous cell carcinoma
Int. J. Oral Maxillofac. Surg. 2008; 37: 372–378 doi:10.1016/j.ijom.2007.11.021, available online at http://www.sciencedirect.com
Research Paper
Expression of vascular endothelial growth factor-C does not predict occult lymphnode metastasis in early oral squamous cell carcinoma
S. E. S. Faustino1, D. T. Oliveira1, S. Nonogaki2, G. Landman3, A. L. Carvalho4, L. P. Kowalski4 1 Department of Stomatology, Area of Pathology, Bauru School of Dentistry University of Sa˜o Paulo, Bauru, Brazil; 2Adolfo Lutz Institute, Pathology Division, Sa˜o Paulo, Brazil; 3Department of Pathology, A. C. Camargo Cancer Hospital, Sa˜o Paulo, Brazil; 4 Department of Head and Neck Surgery and Otorhinolaryngology, A. C. Camargo Cancer Hospital, Sa˜o Paulo, Brazil
S. E. S. Faustino, D. T. Oliveira, S. Nonogaki, G. Landman, A. L. Carvalho, L. P. Kowalski: Expression of vascular endothelial growth factor-C does not predict occult lymph-node metastasis in early oral squamous cell carcinoma. Int. J. Oral Maxillofac. Surg. 2008; 37: 372–378. # 2007 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved. Abstract. Strong vascular endothelial growth factor-C (VEGF-C) expression has been correlated to occurrence of lymph-node metastases in patients with oral squamous cell carcinoma (OSCC). The incidence of occult lymph-node metastasis remains a decisive factor in the prognosis of patients with early OSCC. The aim of this study was to evaluate VEGF-C expression as a predictor of occult lymph-node metastasis in OSCC. Eighty-seven patients with primary OSCC arising in the tongue or floor of mouth, clinically T1N0M0 or T2N0M0, with (pN+) and without (pN0) occult lymph-node metastases were analyzed for VEGF-C expression by malignant cells. Occult lymph-node metastases (pN+) were detected in 22% of the 64 patients who were submitted to elective neck dissection. No statistically significant difference was found between OSCC with and without occult lymph-node metastasis in regard to VEGF-C immunoexpression by malignant cells and clinicopathologic features. Independently of VEGF-C expression, lymph-node metastasis (pN+) was the most significant prognostic factor for overall survival of patients with OSCC (p = 0.030). These findings indicate that isolated VEGF-C expression by malignant cells is not of predictive value for occult lymph-node metastasis in the early stages of OSCC.
Tumor invasion and metastasis is a critical event of human cancer frequently associated with a fatal outcome. It has been speculated that the VEGF family, particularly VEGF-C and VEGF-D, may function 0901-5027/040372 + 07 $30.00/0
as angiogenic/lymphangiogenic factors that facilitate tumor progression and metastasis9,15,23,29,30,43. The role of VEGF-C in angiogenesis/ lymphangiogenesis, disease progression
Keywords: VEGF-C; oral squamous cell carcinoma; occult metastasis. Accepted for publication 26 November 2007 Available online 4 March 2008
and patient prognosis has been extensively studied in various types of human tumor, including OSCC2,4,19,22,24,28,37,42. Evidence is emerging to correlate the presence of VEGF-C expression in tumor
# 2007 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
VEGF-C expression and occult lymph-node metastasis in oral cancer cells with an increased likelihood of lymph-node metastases, and consequently to suggest it as a prognostic indicator in oral cancer11,16,17,20,21,25,33,35,36,41,44. It is well established that positive pathologic neck nodes (pN+) have the most decisive influence on prognosis in patients with OSCC13. The incidence of occult lymph-node metastasis (pN+) in patients with OSCC, clinical stages I and II, ranging from 23.1% to 45%1,7,26,31, continues to be one of the strongest arguments indicating elective neck dissection of these tumors. There are also a high percentage of patients who do not have metastasis in the pathological exam (pN0), and in such cases the undesirable cosmetic and functional effects of neck dissection ideally should be avoided1. Determining whether or not elective neck dissection will be beneficial to the patient continues to be an important clinical dilemma. Detection of better markers to identify patients with biologically aggressive tumors and/or poor prognoses would provide a much-needed opportunity to target patients at risk of development of metastasis24, contributing to the proper selection of patients for elective neck dissection1. Hence, there is an urgent need to identify characteristics of the primary tumor that might predict nodal metastasis36, improving the patient’s survival10,11. In this study, isolated VEGF-C expression by malignant cells as a predictor of occult lymph-node metastasis was investigated in early stages of OSCC located in the tongue and floor of mouth. Patients and Methods Patient and tumor samples
This study was based on the analysis of 87 patients (68 males and 19 females) who underwent surgical treatment for primary OSCC from 1968 to 2001, at the Head and Neck Surgery and Otorhinolaryngology Department of the A.C. Camargo Cancer Hospital, Brazil. The inclusion criteria were: (1) primary OSCC located in the tongue or floor of mouth, clinical stages I (T1N0M0) and II (T2N0M0), confirmed by biopsy; (2) patients who did not undergo radiotherapy, chemotherapy or other treatment prior to surgery; (3) patients without other simultaneous primary tumors; (4) tumor tissue available for microscopic analysis. Clinical data of the patients were obtained from the medical records and included gender, age, ethnic group (white or not white), tobacco and alcohol consumption, tumor location, TNM stage38,39, treatment (surgery, postoperative adjuvant
radiotherapy), and clinical follow up (local recurrence, regional recurrence and death). A formalin-fixed 3-mm section of tumor tissue was taken from the pathology archive for hematoxylin & eosin (HE) staining and immunohistochemistry analyses of occult lymph-node metastasis and VEGF-C expression. The histopathological malignancy grading of the OSCC was established by an experienced pathologist according to BRYNE et al.6 (1989), without knowledge of the clinical data. The presence of vascular embolization, and perineural, muscular and salivary gland infiltrations in the OSCC were reviewed in the HE stained tumor sections. VEGF-C immunoexpression in OSCC
The 87 specimens of OSCC previously fixed in 10% buffered formalin and embedded in paraffin were cut into 3mm-thick sections for the standard streptavidin-biotin–peroxidase complex method (StreptABComplex/HRP, Duet Mouse/Rabbit, Dako ref K0492, Glostrup, Denmark). After antigen retrieval using 10 mM citrate buffer, pH 6.0, in a pressure cooker for 4 min, endogenous peroxidase activity was blocked by incubation in 3% H2O2 for 20 min. The sections were incubated overnight at 4 8C with the primary antibody VEGF-C (C-20) (Santa Cruz Biotechnology, sc-1881, Santa Cruz, CA, USA), dilution 1:100, in phosphatebuffered saline with bovine serum albumin solution to block a non-specific reaction. Then, the tumor sections were incubated with second antibody biotinylated anti-goat Ig made in rabbit (Vector BA-5000, Burlingame, CA, USA) diluted in phosphate-buffered saline 1:500, for 30 min, at 37 8C. The antigen–antibody reaction was detected using streptavidinbiotin-based detection kit StreptABComplex/HRP Duet, mouse/rabbit (Dako A/S, K0492, Denmark) and visualized using 3.3’diaminobenzidine tetrahydrochloride (DAB/SIGMA, ref D-5637, St. Louis, MO, USA). Sections were counterstained with Harris hematoxylin before being dehydrated and cover slipped. Human placenta was used as positive control. Normal oral mucosa from the surgical margins was used as internal control. For a negative control, the primary antibody was omitted during the immunohistochemical staining. Quantitative computer-assisted analysis of 30 invasive tumor front fields (X400 magnification: 93,992.05 mm2/field) in each tumor sample was performed to evaluate the cytoplasmic immunoreactivity of VEGF-C expressed by malignant cells. The images of the invasive tumor front in each
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section were digitally captured with a camera (Axiocam MR3, Zeiss, Jena, Germany) attached to a light microscope (Axioskop2 Plus, Zeiss, Jena, Germany) and recorded by a computer program system (Axiovision 4.5, Zeiss, Jena, Germany). Immunostaining results of VEGF-C expressed by malignant cells were evaluated by two investigators without prior knowledge of the tumor’s histopathologic features and the patient’s clinical status. A combined score for VEGF-C expression was based on: (a) intensity of the immunostaining (0 = negative; 1 = weak; 2 = moderate; 3 = strong; 4 = very strong) and (b) percentage of positive cells (0 = 0% positive cells; 1 = <25% positive cells; 2 = 25–50% positive cells; 3 = 50–75% positive cells; 4 = >75% positive cells), as described previously by SOINI et al.40. The final immunostaining score was determined by the sum of (a) + (b) ranged from 0 to maximally 8. A final score with value greater than 6 was considered as strong VEGF-C expression.
Statistical analysis
All statistical analyses were performed using SPSS statistical software version 10.0 (SPSS Inc., Chicago, IL, USA). Associations between VEGF-C expression and the variables studied were verified according to Chi-square test or Fisher’s exact test. P values less than 0.05 were considered statistically significant. The survival probabilities (overall and disease-free survivals) were estimated using the Kaplan– Meier method and to compare survival curves the log-rank test was used. The follow-up period was the time between surgery date and the death or last patient information date. Results Clinical features
The analysis of 87 patients with OSCC revealed a white male predominance with frequently tobacco (83%) and/or alcohol (76%) consumption. The patients’ ages ranged from 35 to 89 years (mean 59.36 years 10.91 standard deviation [SD]). The tumor was located in the tongue (69% of cases) and floor of the mouth (31% of cases). On the basis of the Union Internationale Contre le Cancer38,39 criteria of oral cavity carcinomas, a total of 28 cases (32%) were at clinical stage I (T1N0M0) and 59 (68%) clinical stage II (T2N0M0). All 87 patients underwent surgical treatment of primary tumor resection and 64 of them were submitted to elective neck dis-
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Fig. 1. Weak cytoplasmic immunostaining for VEGF-C in OSCC (A and B). Strong cytoplasmic immunostaining for VEGF-C in OSCC (C and D).
section (54 patients: ipsilateral neck dissection; 9 patients: bilateral neck dissection). Only 19 patients received postoperative adjuvant radiotherapy. During clinical follow up, local and regional recurrences were observed in 15 (17%) and 14 (16%) patients respectively, and three patients (3.4%) developed both. Two patients with OSCC developed distant metastases (one within lung and the other liver). Microscopically, most of the OSCC cases analysed were well to moderately differentiated, showing solid cords of neoplastic cells as pattern of invasion. Most of the tumors presented intense keratinization, discrete nuclear polymorphism, few mitotic figures per high-power field (X400 magnification) and intense-to-moderate lymphocytic infiltration. According to histopathological malignant grading described by BRYNE et al.6, 69 OSCCs (79%) were classified as more differentiated tumors (5–12 points) and 18 (21%) as less differentiated tumors (13–20 points). Surgical margins were negative in 84 patients (97%), but three patients (3.4%) had positive surgical margins (a surgical margin was classified as positive when there was OSCC or OSCC in situ at the margin). Fourteen patients (22%) among the 64 patients submitted to
elective neck dissection presented positive lymph nodes confirmed by histopathological analysis (pN+) at the moment of primary tumor resection. Immunostaining for VEGF-C expressed by malignant cells
Immunohistochemical analysis showed strong cytoplasmic immunostaining for VEGF-C in 66 (75.9%) of the 87 tumors and weak immunostaining in 21 (24%) (Fig. 1). Some of the inflammatory cells infiltrating the tumors, especially macrophages, were found to stain strongly for VEGF-C. No statistically significant correlations were found regarding clinicopathologic parameters (including gender, ethnic group, age, tobacco, alcohol, tumor site, T stage, local recurrence, regional recurrence, lymphatic or blood embolization, perineural, muscular and salivary gland infiltrations) and the VEGF-C expression by malignant cells of the OSCC (Table 1). There was no statistical correlation between VEGF-C expression and occult lymph-node metastases as well as histopathological malignancy grading in the invasive front of the tumors (Table 2). The clinical follow up for the 87 patients with OSCC ranged from 5.4 to
272.1 months (mean 82.2 63.1 SD). At the end of the follow-up period, 33 patients (38%) were alive and disease free, 19 patients (22%) had died of recurrence (local, regional or distant), 28 patients (32%) had died from cause other than tumor, and 7 (8%) were considered lost to follow up because they reached a clinical outcome in less than 5 years. Details concerning 5-year and 10-year overall and disease-free survival rates are summarized in Table 3. VEGF-C expression by malignant cells was not a significant prognostic factor for patients with OSCC. Excluding VEGF-C expression, the neck lymph-nodal status influenced the overall survival rates of the 64 patients submitted to elective neck dissection (Fig. 2 and Table 4). The 5-year and 10year overall survival rates were, respectively, 67% and 58% (pN0 group), whereas for the 14 patients with occult lymph-node metastasis they were 50% and 29% (pN+ group) (p = 0.030), as summarized in Table 4. Discussion
Although the metastatic dissemination of tumor cells to regional lymph nodes is a common feature of OSCC arising in the
VEGF-C expression and occult lymph-node metastasis in oral cancer Table 1. Correlation between clinicopathologic parameters and VEGF-C expression in OSCC VEGF-C expression Weak
P
Strong
Variable
Category
N
%
N
%
Gender
Male Female White Not white 59 years >59 years Yes No Yes No Tongue Floor of mouth T1 T2 Yes No Yes No Yes No Yes No Yes No Yes No Yes No
16 5 20 1 10 11 17 3 16 4 16 5 10 11 2 19 4 17 5 16 2 19 12 9 18 3 5 16 21
76 24 95 5 48 52 85 15 80 20 76 24 48 52 10 90 19 81 24 76 10 90 57 43 86 14 24 76 100
52 14 60 6 35 31 55 6 50 11 44 22 18 48 13 53 10 56 20 46 10 56 32 34 53 13 24 42 66
79 21 91 9 53 47 90 10 82 18 67 33 27 73 20 80 15 85 30 70 15 85 49 51 80 20 36 64 100
Ethnic group Age Tobacco
*
Alcohol* Tumor site T stage Local recurrence Regional recurrence Lymphatic embolization Blood embolization Perineural infiltration Muscular infiltration Salivary gland infiltration TOTAL *
0.802 0.525 0.666 0.524 0.844 0.411 0.082 0.282 0.672 0.567 0.515 0.489 0.577 0.288
Excluding patients with lost records.
sels9,15,23,29,30,43, but little is known about the role of tumor lymphangiogenesis in metastasis. VEGF-C overexpression seems to induce new lymphatic and blood vessels in the vicinity of the primary cancer14,33. In this context, the correlation between VEGF-C and lymphatic vessel density in human OSCC, evaluated by immunohistochemical parameters25,33,36,
tongue and floor of mouth, it is not clear whether the tumor utilizes existing lymphatic channels or whether dissemination requires the formation of new lymphatic vessels (lymphangiogenesis)4,8,18,27,32,34. In the last decade, VEGF-C and VEGFD were identified as lymphangiogenic growth factors and ligands to the receptor VEGFR-3 present in the lymphatic ves-
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has gained importance in recent years as an adjuvant tool, in order to understand the mechanism of metastatic spread via lymphatic vessels. Most of the patients with OSCC submitted to elective neck dissection in our series showed strong cytoplasmic immunostaining for VEGF-C, including pN0 and those with occult lymph node metastasis (pN+), as shown in Fig. 1. These results confirm the observation that tumor cells of OSCC, in the early stage of development, express VEGF-C11,16,17,20,21,25,33,35,36,41,44, which may elevate the risk of precocious nodal metastasis. VEGF-C expression of OSCC located in the tongue and floor of mouth was not influenced by other clinicopathological parameters (gender, ethnic group, age, tobacco and alcohol consumption, tumor site, T stage, local recurrence, regional recurrence, lymphatic or blood embolization, perineural, muscular and salivary gland infiltration) as described in Table 1. In contrast to the results of KISHI11 16 33 MOTO et al. , LI et al. , SEDIVY et al. , 35 36 SHINTANI et al. , SIRIWARDENA et al. and TANIGAKI et al.41, the present results do not show a statistically significant association between lymph-node metastasis and VEGF-C expression by malignant cells of the OSCC (clinical stage I and II). These authors did not investigate, as in the present study, the occult lymph-node metastasis from OSCC, and most of them analyzed tumors at both early and advanced clinical stages (from I to IV). Occult lymph-node metastases (pN+) were detected in 22% of the 64 patients who were submitted to elective neck dissection in this series. This percentage is close to those reported by AMARAL et al.1 (23%) and OKAMOTO et al.26 (24%), but
Table 2. Correlation between histopathological malignancy grading, neck lymph-nodal status and VEGF-C expression in OSCC VEGF-C Weak Variable Malignancy grading Neck lymph-nodal status*
P
Strong
TOTAL
Category
N
%
N
%
N
%
Less differentiated More differentiated pN+ (ONM) pN0
3 18 2 8
17 26 14 16
15 51 12 42
83 74 86 84
18 69 14 50
100 100 100 100
0.406 0.876
ONM = occult lymph node metastasis. * Excluding patients not submitted to elective neck dissection. Table 3. Five-year and 10-year survival rates of the 87 patients with OSCC according to VEGF-C expression Variable VEGF-C
Category Weak Strong
Overall survival (%) 5-year
10-year
63 62
53 46
P 0.682
Disease-free survival (%) 5-year
10-year
65 66
65 66
P 0.975
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Table 4. Five-year and 10-year overall survival rates of the 64 patients with OSCC submitted to elective neck dissection Variable Neck lymph-nodal status
Category pN+ (ONM) pN0
Overall survival (%) 5-year
10-year
50 67
29 58
P 0.030
ONM = occult lymph-node metastasis.
lower than those of RUSSOLO et al.31 (34%) and BYERS et al.7 (45%) in patients with OSCC. The immunostaining of VEGF-C in OSCC is a histopathological parameter that has been evaluated for others11,17,33,35,41 but generally few microscopic fields were analyzed and frequently the extension of the examined area and field magnification were not specified. The present study involved an objective and reproducible evaluation of VEGF-C expression in malignant cells of OSCC using a computer system that permitted capture of the tumor area to be analyzed by investigators. Thirty invasive front tumor fields were evaluated (X400 magnification) in each sample; without doubt, a representative area of the most invasive region of the OSCC. Use of this objective methodology minimizing the subjectivity and facilitates comparison of the results with those of future studies, an essential step for establishment of the real influence of VEGF-C expression by malignant cells on the clinical and biological behavior of the OSCC. According to the histopathological malignancy grading, most (79%) of the 87 OSCC cases were classified as well-tomoderately differentiated (more differentiated tumors), a finding consistent with other published studies11,35,41. There was no statistical correlation between malig-
nancy grading and VEGF-C expression (Table 2). Bryne’s grading system6 was used, in which the morphological features are determined from the most invasive front of the tumors. The invasive tumor front consists of the most aggressive cells, which have the ability to invade surrounding tissue structures, including vessels, and thereby metastasize. The characteristics of the invasive tumor front are of major significance for the prognosis of oral cancer3,5. VEGF-C expression in the malignant cells did not influence the likelihood of overall and disease-free survival for the patients with OSCC located in the tongue and floor of the mouth (Table 3). The only prognostic factor that reached statistical significance and influenced cumulative overall survival rates was the occurrence of occult lymph-node metastasis (pN+), as showed in Table 4 and Fig. 2. These results reinforce previous findings that, in the early stages of OSCC, the prediction of occult lymph-node metastasis is, at present, indispensable for improvement in the probability of survival1,7,10,11,12,31. The pursuit of predictive factors should be increased to reduce the morbidity of elective neck dissection in patients who do not have metastasis in the pathological exam (pN0). In conclusion, although VEGF-C expression has been occasionally regarded
Fig. 2. Cumulative overall survival probability curves for groups with lymph-node metastasis (pN+) and without lymph-node metastasis (pN0).
as a prognostic factor in OSCC, these results suggest that the isolated immunoexpression of VEGF-C in malignant cells does not directly influence the clinical outcome and prognosis of patients with early OSCC of tongue and floor of mouth. In spite of this, further studies should be performed with regard to VEGF-C, microvessel density and the occurrence of regional lymph-node metastases because, in the future, understanding the influence of lymphangiogenic growth factors and tumour immunomodulation may yield an array of new therapies to limit the metastatic spread of oral cancer. Acknowledgments. The authors thank FAPESP (Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo, grant #2005/ 04577-4) and CAPES (Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior) for supporting this study.
References 1. Amaral TMP, Freire ARS, Carvalho AL, Pinto CAL, Kowalski LP. Predictive factors of occult metastasis and prognosis of clinical stages I and II squamous cell carcinoma of the tongue and floor of the mouth. Oral Oncol 2004: 40: 780– 786. 2. Arinaga M, Noguchi T, Takeno S, Chujo M, Miura T, Uchida Y. Clinical significance of vascular endothelial growth factor C and vascular endothelial growth factor receptor 3 in patients with nonsmall cell lung carcinoma. Cancer 2003: 97: 457–464. 3. Ba`nkfalvi A, Piffko` J. Prognostic and predictive factors in oral cancer: the role of invasive tumor front. J Oral Path Med 2000: 29: 291–298. 4. Beasley NJP, Prevo R, Banerji S, Leek RD, Moore J, van Trappen P, Cox G, Harris AL, Jackson DG. Intratumoral lymphangiogenesis and lymph node metastasis in head and neck cancer. Cancer Res 2002: 62: 1315–1320. 5. Bryne M, Boysen M, Alfsen CG, Abeler VM, Sudbo J, Nesland JM, Kristensen GB, Piffko J, Bankfalvi A. The invasive front of carcinomas. The most important area for tumour prognosis? Anticancer Res 1998: 18: 4757–4764. 6. Bryne M, Koppang HS, Lilleng R, Stene T, Bang G, Dabelsteen E. New malignancy grading is a better prognostic indicator than Broders’ grading in oral squamous cell carcinoma. J Oral Pathol Med 1989: 18: 432–437. 7. Byers RM, El-Naggar AK, Lee YY, Rao B, Fornage B, Terry NH, Sample D, Hankins P, Smith TL, Wolf PJ. Can we detect or predict the presence of occult nodal metastases in patients with
VEGF-C expression and occult lymph-node metastasis in oral cancer
8.
9. 10.
11.
12.
13. 14.
15.
16.
17.
18.
19.
squamous carcinoma of the oral tongue? Head Neck 1998: 20: 138–144. Clarijs R, Ruiter DJ, De Wall RM. Lymphangiogenesis in malignant tumours: does it occur? J Pathol 2001: 193: 143–146. Ferrara N, Davis-Smyth T. The biology of vascular endothelial growth factor. Endocr Rev 1997: 18: 4–25. Haddadin KJ, Soutar DS, Oliver RJ, Webster MH, Robertson AG, MacDonald DG. Improved survival for patients with clinically T1/T2, N0 tongue tumors undergoing a prophylactic neck dissection. Head Neck 1999: 21: 517–525. Kishimoto K, Sasaki A, Yoshihama Y, Mese H. Expression of vascular endothelial growth factor-C predicts regional lymph node metastasis in early oral squamous cell carcinoma. Oral Oncol 2003: 39: 391–396. KOWALSKI LP, MAGRIN J, SAB_OIA M, SANTOS JC, TORLONI H. Squamous cell carcinoma of the tongue: a review of 629 patients treated at a single institution. South Am J Cancer 1997: 1: 245-254. Kowalski LP, Medina JE. Nodal metastases: predictive factors. Otolaryngol Clin North Am 1998: 31: 621–637. Krishnan J, Kirkin V, Steffen A, Hegen M, Weih D, Tomarev S, Wilting J, Sleeman JP. Differential in vivo and in vitro expression of vascular endothelial growth factor (VEGF)-C and VEGF-D in tumors and its relationship to lymphatic metastasis in immunocompetent rats. Cancer Res 2003: 63: 713–722. Kukk E, Lymboussaki. Taira S, Kaipanen A, Jeltsch M, Joukov V, Alitalo K. VEGF-C receptor binding, and pattern of expression with VEGFR-3 suggests a role in lymphatic vascular development. Development 1996: 122: 3829– 3837. Li QL, Chen FJ, Zeng ZY, Yang AK, Wu QL, Zhang HZ, Hou JH. Expression and clinical significance of VEGF-C and Flt-4 in tongue squamous cell carcinoma. Ai Zheng 2006: 25: 235–240. Lo´pez De Cicco R, Watson JC, Bassi DE, Litwin S, Klein-Szanto1 AJ. Simultaneous expression of furin and vascular endothelial growth factor in human oral tongue squamous cell carcinoma progression. Clin Cancer Res 2004: 10: 4480–4488. Munoz-Guerra MF, Marazuela EG, Martin-Villar E, Quintanilla M, Gamallo C. Prognostic significance of intratumoral lymphangiogenesis in squamous cell carcinoma of the oral cavity. A study of the early stages. Cancer 2004: 100: 553–560. Nakamura Y, Yasuoka H, Tsujimoto M, Yang Q, Tsukiyama A, Imabun S, Nakahara M, Nakao K, Nakamura M, Mori I, Kakudo K. Clinicopathological significance of vascular endothelial growth factor-C in breast carcinoma with
20.
21.
22.
23.
24.
25.
26.
27. 28.
29.
30.
31.
long-term follow-up. Mod Pathol 2003: 16: 309–314. Nakaya H, Kawashiri S, Tanaka A, Noguchi N, Kato K, Hase T, Yamamoto E. Influences of angiogenesis and lymphangiogenesis on cancerous invasion in experimentally induced tongue carcinoma. J Oral Pathol Med 2005: 34: 87–92. Nakazato T, Shingaki S, Kitamura N, Saito C, Kuwano R, Tachibana M. Expression level of vascular endothelial growth factor-C and -A in cultured human oral squamous cell carcinoma correlates respectively with lymphatic metastasis and angiogenesis when transplanted into nude mouse oral cavity. Oncol Rep 2006: 15: 825–830. Neuchrist C, Erovic BM, Handisurya A, Fischer MB, Steiner GE, Hollemann D, Gedlicka C, Saaristo A, Burian M. Vascular endothelial growth factor C and vascular endothelial growth factor receptor 3 expression in squamous cell carcinomas of the head and neck. Head Neck 2003: 25: 464–474. Nicosia RF. What is the role of vascular endothelial growth factor-related molecules in tumor angiogenesis? Am J Pathol 1998: 153: 11–16. O-Charoenrat P, Rhys-Evans P, Eccles SA. Expression of vascular endothelial growth factor family members in head and neck squamous cell carcinoma correlates with lymph node metastasis. Cancer 2001: 92: 556–568. Ohno F, Nakanishi H, Abe A, Seki Y, Kinoshita A, Hasegawa Y, Tatematsu M, Kurita K. Regional difference in intratumoral lymphangiogenesis of oral squamous cell carcinomas evaluated by immunohistochemistry using D240 and podoplanin antibody: an analysis in comparison with angiogenesis. J Oral Pathol Med 2007: 36: 281–289. Okamoto M, Nishimine M, Kishi M, Kirita T, Sugimura M, Nakamura M, Konishi N. Prediction of delayed neck metastasis in patients with stage I/ II squamous cell carcinoma of the tongue. J Oral Pathol Med 2002: 31(4):227–233. Pepper MS. Lymphangiogenesis and tumor metastasis: myth or reality? Clin Cancer Res 2001: 7: 462–468. Pepper MS, Tille JC, Nisato R, Skobe M. Lymphangiogenesis and tumor metastasis. Cell Tissue Res 2003: 314: 167– 177. Ribatti D, Nico B, Crivellato E, Roccaro AM, Vacca A. The history of the angiogenic switch concept. Leukemia 2007: 21: 44–52. Roskoski Jr R. Vascular endothelial growth factor (VEGF) signaling in tumor progression. Crit Rev Oncol Hematol 2007: 62: 179–213 Epub 2007 Feb 26.. Russolo M, Giacomarra V, Papanikolla L, Tirelli G. Prognostic indicators of occult metastases in oral cancer. Laryngoscope 2002: 112: 1320–1323.
377
32. Saharinen P, Tammela T, Karkkainen MJ, Alitalo K. Lymphatic vasculature: development, molecular regulation and role in tumor metastasis and inflammation. Trends Immunol 2004: 25: 387–395. 33. Sedivy R, Beck-Mannagetta J, Haverkampf C, Battistutti W, Ho¨nigschnabl S. Expression of vascular endothelial growth factor-C correlates with the lymphatic microvessel density and the nodal status in oral squamous cell cancer. J Oral Pathol Med 2003: 32: 455– 460. 34. Shayan R, Achen MG, Stacker SA. Lymphatic vessels in cancer metastasis: bridging the gaps. Carcinogenesis 2006: 27: 1729–1738. 35. Shintani S, Li C, Ishikawa T, Mihara M, Nakashiro K, Hamakawa H. Expression of vascular endothelial growth factor A, B, C and D in oral squamous cell carcinoma. Oral Oncol 2004: 40: 13–20. 36. Siriwardena BSMS, Kudo Y, Ogawa I, Udagama M.N.G.P.K.. Tilakaratne WM, Takata T. VEGF-C is associated with lymphatic status and invasion in oral cancer. J Clin Pathol 2007: 9 [Epub ahead of print].. 37. Skobe M, Hawighorst T, Jackson DG, Prevo R, Janes L, Velasco P, Riccardi L, Alitalo K, Claffey K, Detmar M. Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis. Nat Med 2001: 7: 192– 198. 38. Sobin LH, Fleming ID. TNM classification of malignant tumors, fifth edition. Union Internationale Contre le Cancer and the American Joint Committee on Cancer. Cancer 1997: 80: 1803–1804. 39. Sobin LH, Wittekind CH. TNM: classification of malignant tumors. 6th ed. New York: John Wiley & Sons 2002. 40. Soini Y, Puhakka A, Kahlos K, Sa¨ily M, Pa¨a¨kko¨ P, Koistinen P, Kinnula V. Endothelial nitric oxide synthase is strongly expressed in malignant mesothelioma but does not associate with vascular density or the expression of VEGF, Flk1 or Flt1. Histopathology 2001: 39: 179– 186. 41. Tanigaki Y, Nagashima Y, Kitamura Y, Matsuda H, Mikami Y, Tsukuda M. The expression of vascular endothelial growth factor –A and –C, and receptors 1 and 3: Correlation with lymph node metastasis and prognosis in tongue squamous cell carcinoma. Inter J Mol Med 2004: 14: 389–395. 42. Tsurusaki T, Kanda S, Sakai H, Kanetake H, Saito Y, Alitalo K, Koji T. Vascular endothelial growth factor-C expression in human prostatic carcinoma and its relationship to lymph node metastasis. Br J Cancer 1999: 80: 309– 313. 43. Veikkola T, Karkkainen M, Claesson-Welsh L, Alitalo K. Regulation
378
Faustino et al.
of angiogenesis via vascular endothelial growth factor receptors. Cancer Res 2000: 60: 203–212. 44. Warburton G, Nikitakis NG, Roberson P, Marinos NJ, Wu T, Sauk JJ, Ord RA, Wahl SM. Histopathological and lymphangiogenic parameters in relation to lymph node metastasis in early stage
oral squamous cell carcinoma. J Oral Maxilofac Surg 2007: 65: 475–484. Address: Denise Tostes Oliveira Faculdade de Odontologia de Bauru A´rea de Patologia Alameda Octa´vio Pinheiro Brisolla
9-75 CEP 17012-901 Bauru Sa˜o Paulo Brazil Tel.: +55 21 14 32358251 Fax: +55 21 14 32234679 E-mail: [email protected]
Research Paper
Expression of vascular endothelial growth factor-C does not predict occult lymphnode metastasis in early oral squamous cell carcinoma
S. E. S. Faustino1, D. T. Oliveira1, S. Nonogaki2, G. Landman3, A. L. Carvalho4, L. P. Kowalski4 1 Department of Stomatology, Area of Pathology, Bauru School of Dentistry University of Sa˜o Paulo, Bauru, Brazil; 2Adolfo Lutz Institute, Pathology Division, Sa˜o Paulo, Brazil; 3Department of Pathology, A. C. Camargo Cancer Hospital, Sa˜o Paulo, Brazil; 4 Department of Head and Neck Surgery and Otorhinolaryngology, A. C. Camargo Cancer Hospital, Sa˜o Paulo, Brazil
S. E. S. Faustino, D. T. Oliveira, S. Nonogaki, G. Landman, A. L. Carvalho, L. P. Kowalski: Expression of vascular endothelial growth factor-C does not predict occult lymph-node metastasis in early oral squamous cell carcinoma. Int. J. Oral Maxillofac. Surg. 2008; 37: 372–378. # 2007 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved. Abstract. Strong vascular endothelial growth factor-C (VEGF-C) expression has been correlated to occurrence of lymph-node metastases in patients with oral squamous cell carcinoma (OSCC). The incidence of occult lymph-node metastasis remains a decisive factor in the prognosis of patients with early OSCC. The aim of this study was to evaluate VEGF-C expression as a predictor of occult lymph-node metastasis in OSCC. Eighty-seven patients with primary OSCC arising in the tongue or floor of mouth, clinically T1N0M0 or T2N0M0, with (pN+) and without (pN0) occult lymph-node metastases were analyzed for VEGF-C expression by malignant cells. Occult lymph-node metastases (pN+) were detected in 22% of the 64 patients who were submitted to elective neck dissection. No statistically significant difference was found between OSCC with and without occult lymph-node metastasis in regard to VEGF-C immunoexpression by malignant cells and clinicopathologic features. Independently of VEGF-C expression, lymph-node metastasis (pN+) was the most significant prognostic factor for overall survival of patients with OSCC (p = 0.030). These findings indicate that isolated VEGF-C expression by malignant cells is not of predictive value for occult lymph-node metastasis in the early stages of OSCC.
Tumor invasion and metastasis is a critical event of human cancer frequently associated with a fatal outcome. It has been speculated that the VEGF family, particularly VEGF-C and VEGF-D, may function 0901-5027/040372 + 07 $30.00/0
as angiogenic/lymphangiogenic factors that facilitate tumor progression and metastasis9,15,23,29,30,43. The role of VEGF-C in angiogenesis/ lymphangiogenesis, disease progression
Keywords: VEGF-C; oral squamous cell carcinoma; occult metastasis. Accepted for publication 26 November 2007 Available online 4 March 2008
and patient prognosis has been extensively studied in various types of human tumor, including OSCC2,4,19,22,24,28,37,42. Evidence is emerging to correlate the presence of VEGF-C expression in tumor
# 2007 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
VEGF-C expression and occult lymph-node metastasis in oral cancer cells with an increased likelihood of lymph-node metastases, and consequently to suggest it as a prognostic indicator in oral cancer11,16,17,20,21,25,33,35,36,41,44. It is well established that positive pathologic neck nodes (pN+) have the most decisive influence on prognosis in patients with OSCC13. The incidence of occult lymph-node metastasis (pN+) in patients with OSCC, clinical stages I and II, ranging from 23.1% to 45%1,7,26,31, continues to be one of the strongest arguments indicating elective neck dissection of these tumors. There are also a high percentage of patients who do not have metastasis in the pathological exam (pN0), and in such cases the undesirable cosmetic and functional effects of neck dissection ideally should be avoided1. Determining whether or not elective neck dissection will be beneficial to the patient continues to be an important clinical dilemma. Detection of better markers to identify patients with biologically aggressive tumors and/or poor prognoses would provide a much-needed opportunity to target patients at risk of development of metastasis24, contributing to the proper selection of patients for elective neck dissection1. Hence, there is an urgent need to identify characteristics of the primary tumor that might predict nodal metastasis36, improving the patient’s survival10,11. In this study, isolated VEGF-C expression by malignant cells as a predictor of occult lymph-node metastasis was investigated in early stages of OSCC located in the tongue and floor of mouth. Patients and Methods Patient and tumor samples
This study was based on the analysis of 87 patients (68 males and 19 females) who underwent surgical treatment for primary OSCC from 1968 to 2001, at the Head and Neck Surgery and Otorhinolaryngology Department of the A.C. Camargo Cancer Hospital, Brazil. The inclusion criteria were: (1) primary OSCC located in the tongue or floor of mouth, clinical stages I (T1N0M0) and II (T2N0M0), confirmed by biopsy; (2) patients who did not undergo radiotherapy, chemotherapy or other treatment prior to surgery; (3) patients without other simultaneous primary tumors; (4) tumor tissue available for microscopic analysis. Clinical data of the patients were obtained from the medical records and included gender, age, ethnic group (white or not white), tobacco and alcohol consumption, tumor location, TNM stage38,39, treatment (surgery, postoperative adjuvant
radiotherapy), and clinical follow up (local recurrence, regional recurrence and death). A formalin-fixed 3-mm section of tumor tissue was taken from the pathology archive for hematoxylin & eosin (HE) staining and immunohistochemistry analyses of occult lymph-node metastasis and VEGF-C expression. The histopathological malignancy grading of the OSCC was established by an experienced pathologist according to BRYNE et al.6 (1989), without knowledge of the clinical data. The presence of vascular embolization, and perineural, muscular and salivary gland infiltrations in the OSCC were reviewed in the HE stained tumor sections. VEGF-C immunoexpression in OSCC
The 87 specimens of OSCC previously fixed in 10% buffered formalin and embedded in paraffin were cut into 3mm-thick sections for the standard streptavidin-biotin–peroxidase complex method (StreptABComplex/HRP, Duet Mouse/Rabbit, Dako ref K0492, Glostrup, Denmark). After antigen retrieval using 10 mM citrate buffer, pH 6.0, in a pressure cooker for 4 min, endogenous peroxidase activity was blocked by incubation in 3% H2O2 for 20 min. The sections were incubated overnight at 4 8C with the primary antibody VEGF-C (C-20) (Santa Cruz Biotechnology, sc-1881, Santa Cruz, CA, USA), dilution 1:100, in phosphatebuffered saline with bovine serum albumin solution to block a non-specific reaction. Then, the tumor sections were incubated with second antibody biotinylated anti-goat Ig made in rabbit (Vector BA-5000, Burlingame, CA, USA) diluted in phosphate-buffered saline 1:500, for 30 min, at 37 8C. The antigen–antibody reaction was detected using streptavidinbiotin-based detection kit StreptABComplex/HRP Duet, mouse/rabbit (Dako A/S, K0492, Denmark) and visualized using 3.3’diaminobenzidine tetrahydrochloride (DAB/SIGMA, ref D-5637, St. Louis, MO, USA). Sections were counterstained with Harris hematoxylin before being dehydrated and cover slipped. Human placenta was used as positive control. Normal oral mucosa from the surgical margins was used as internal control. For a negative control, the primary antibody was omitted during the immunohistochemical staining. Quantitative computer-assisted analysis of 30 invasive tumor front fields (X400 magnification: 93,992.05 mm2/field) in each tumor sample was performed to evaluate the cytoplasmic immunoreactivity of VEGF-C expressed by malignant cells. The images of the invasive tumor front in each
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section were digitally captured with a camera (Axiocam MR3, Zeiss, Jena, Germany) attached to a light microscope (Axioskop2 Plus, Zeiss, Jena, Germany) and recorded by a computer program system (Axiovision 4.5, Zeiss, Jena, Germany). Immunostaining results of VEGF-C expressed by malignant cells were evaluated by two investigators without prior knowledge of the tumor’s histopathologic features and the patient’s clinical status. A combined score for VEGF-C expression was based on: (a) intensity of the immunostaining (0 = negative; 1 = weak; 2 = moderate; 3 = strong; 4 = very strong) and (b) percentage of positive cells (0 = 0% positive cells; 1 = <25% positive cells; 2 = 25–50% positive cells; 3 = 50–75% positive cells; 4 = >75% positive cells), as described previously by SOINI et al.40. The final immunostaining score was determined by the sum of (a) + (b) ranged from 0 to maximally 8. A final score with value greater than 6 was considered as strong VEGF-C expression.
Statistical analysis
All statistical analyses were performed using SPSS statistical software version 10.0 (SPSS Inc., Chicago, IL, USA). Associations between VEGF-C expression and the variables studied were verified according to Chi-square test or Fisher’s exact test. P values less than 0.05 were considered statistically significant. The survival probabilities (overall and disease-free survivals) were estimated using the Kaplan– Meier method and to compare survival curves the log-rank test was used. The follow-up period was the time between surgery date and the death or last patient information date. Results Clinical features
The analysis of 87 patients with OSCC revealed a white male predominance with frequently tobacco (83%) and/or alcohol (76%) consumption. The patients’ ages ranged from 35 to 89 years (mean 59.36 years 10.91 standard deviation [SD]). The tumor was located in the tongue (69% of cases) and floor of the mouth (31% of cases). On the basis of the Union Internationale Contre le Cancer38,39 criteria of oral cavity carcinomas, a total of 28 cases (32%) were at clinical stage I (T1N0M0) and 59 (68%) clinical stage II (T2N0M0). All 87 patients underwent surgical treatment of primary tumor resection and 64 of them were submitted to elective neck dis-
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Fig. 1. Weak cytoplasmic immunostaining for VEGF-C in OSCC (A and B). Strong cytoplasmic immunostaining for VEGF-C in OSCC (C and D).
section (54 patients: ipsilateral neck dissection; 9 patients: bilateral neck dissection). Only 19 patients received postoperative adjuvant radiotherapy. During clinical follow up, local and regional recurrences were observed in 15 (17%) and 14 (16%) patients respectively, and three patients (3.4%) developed both. Two patients with OSCC developed distant metastases (one within lung and the other liver). Microscopically, most of the OSCC cases analysed were well to moderately differentiated, showing solid cords of neoplastic cells as pattern of invasion. Most of the tumors presented intense keratinization, discrete nuclear polymorphism, few mitotic figures per high-power field (X400 magnification) and intense-to-moderate lymphocytic infiltration. According to histopathological malignant grading described by BRYNE et al.6, 69 OSCCs (79%) were classified as more differentiated tumors (5–12 points) and 18 (21%) as less differentiated tumors (13–20 points). Surgical margins were negative in 84 patients (97%), but three patients (3.4%) had positive surgical margins (a surgical margin was classified as positive when there was OSCC or OSCC in situ at the margin). Fourteen patients (22%) among the 64 patients submitted to
elective neck dissection presented positive lymph nodes confirmed by histopathological analysis (pN+) at the moment of primary tumor resection. Immunostaining for VEGF-C expressed by malignant cells
Immunohistochemical analysis showed strong cytoplasmic immunostaining for VEGF-C in 66 (75.9%) of the 87 tumors and weak immunostaining in 21 (24%) (Fig. 1). Some of the inflammatory cells infiltrating the tumors, especially macrophages, were found to stain strongly for VEGF-C. No statistically significant correlations were found regarding clinicopathologic parameters (including gender, ethnic group, age, tobacco, alcohol, tumor site, T stage, local recurrence, regional recurrence, lymphatic or blood embolization, perineural, muscular and salivary gland infiltrations) and the VEGF-C expression by malignant cells of the OSCC (Table 1). There was no statistical correlation between VEGF-C expression and occult lymph-node metastases as well as histopathological malignancy grading in the invasive front of the tumors (Table 2). The clinical follow up for the 87 patients with OSCC ranged from 5.4 to
272.1 months (mean 82.2 63.1 SD). At the end of the follow-up period, 33 patients (38%) were alive and disease free, 19 patients (22%) had died of recurrence (local, regional or distant), 28 patients (32%) had died from cause other than tumor, and 7 (8%) were considered lost to follow up because they reached a clinical outcome in less than 5 years. Details concerning 5-year and 10-year overall and disease-free survival rates are summarized in Table 3. VEGF-C expression by malignant cells was not a significant prognostic factor for patients with OSCC. Excluding VEGF-C expression, the neck lymph-nodal status influenced the overall survival rates of the 64 patients submitted to elective neck dissection (Fig. 2 and Table 4). The 5-year and 10year overall survival rates were, respectively, 67% and 58% (pN0 group), whereas for the 14 patients with occult lymph-node metastasis they were 50% and 29% (pN+ group) (p = 0.030), as summarized in Table 4. Discussion
Although the metastatic dissemination of tumor cells to regional lymph nodes is a common feature of OSCC arising in the
VEGF-C expression and occult lymph-node metastasis in oral cancer Table 1. Correlation between clinicopathologic parameters and VEGF-C expression in OSCC VEGF-C expression Weak
P
Strong
Variable
Category
N
%
N
%
Gender
Male Female White Not white 59 years >59 years Yes No Yes No Tongue Floor of mouth T1 T2 Yes No Yes No Yes No Yes No Yes No Yes No Yes No
16 5 20 1 10 11 17 3 16 4 16 5 10 11 2 19 4 17 5 16 2 19 12 9 18 3 5 16 21
76 24 95 5 48 52 85 15 80 20 76 24 48 52 10 90 19 81 24 76 10 90 57 43 86 14 24 76 100
52 14 60 6 35 31 55 6 50 11 44 22 18 48 13 53 10 56 20 46 10 56 32 34 53 13 24 42 66
79 21 91 9 53 47 90 10 82 18 67 33 27 73 20 80 15 85 30 70 15 85 49 51 80 20 36 64 100
Ethnic group Age Tobacco
*
Alcohol* Tumor site T stage Local recurrence Regional recurrence Lymphatic embolization Blood embolization Perineural infiltration Muscular infiltration Salivary gland infiltration TOTAL *
0.802 0.525 0.666 0.524 0.844 0.411 0.082 0.282 0.672 0.567 0.515 0.489 0.577 0.288
Excluding patients with lost records.
sels9,15,23,29,30,43, but little is known about the role of tumor lymphangiogenesis in metastasis. VEGF-C overexpression seems to induce new lymphatic and blood vessels in the vicinity of the primary cancer14,33. In this context, the correlation between VEGF-C and lymphatic vessel density in human OSCC, evaluated by immunohistochemical parameters25,33,36,
tongue and floor of mouth, it is not clear whether the tumor utilizes existing lymphatic channels or whether dissemination requires the formation of new lymphatic vessels (lymphangiogenesis)4,8,18,27,32,34. In the last decade, VEGF-C and VEGFD were identified as lymphangiogenic growth factors and ligands to the receptor VEGFR-3 present in the lymphatic ves-
375
has gained importance in recent years as an adjuvant tool, in order to understand the mechanism of metastatic spread via lymphatic vessels. Most of the patients with OSCC submitted to elective neck dissection in our series showed strong cytoplasmic immunostaining for VEGF-C, including pN0 and those with occult lymph node metastasis (pN+), as shown in Fig. 1. These results confirm the observation that tumor cells of OSCC, in the early stage of development, express VEGF-C11,16,17,20,21,25,33,35,36,41,44, which may elevate the risk of precocious nodal metastasis. VEGF-C expression of OSCC located in the tongue and floor of mouth was not influenced by other clinicopathological parameters (gender, ethnic group, age, tobacco and alcohol consumption, tumor site, T stage, local recurrence, regional recurrence, lymphatic or blood embolization, perineural, muscular and salivary gland infiltration) as described in Table 1. In contrast to the results of KISHI11 16 33 MOTO et al. , LI et al. , SEDIVY et al. , 35 36 SHINTANI et al. , SIRIWARDENA et al. and TANIGAKI et al.41, the present results do not show a statistically significant association between lymph-node metastasis and VEGF-C expression by malignant cells of the OSCC (clinical stage I and II). These authors did not investigate, as in the present study, the occult lymph-node metastasis from OSCC, and most of them analyzed tumors at both early and advanced clinical stages (from I to IV). Occult lymph-node metastases (pN+) were detected in 22% of the 64 patients who were submitted to elective neck dissection in this series. This percentage is close to those reported by AMARAL et al.1 (23%) and OKAMOTO et al.26 (24%), but
Table 2. Correlation between histopathological malignancy grading, neck lymph-nodal status and VEGF-C expression in OSCC VEGF-C Weak Variable Malignancy grading Neck lymph-nodal status*
P
Strong
TOTAL
Category
N
%
N
%
N
%
Less differentiated More differentiated pN+ (ONM) pN0
3 18 2 8
17 26 14 16
15 51 12 42
83 74 86 84
18 69 14 50
100 100 100 100
0.406 0.876
ONM = occult lymph node metastasis. * Excluding patients not submitted to elective neck dissection. Table 3. Five-year and 10-year survival rates of the 87 patients with OSCC according to VEGF-C expression Variable VEGF-C
Category Weak Strong
Overall survival (%) 5-year
10-year
63 62
53 46
P 0.682
Disease-free survival (%) 5-year
10-year
65 66
65 66
P 0.975
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Table 4. Five-year and 10-year overall survival rates of the 64 patients with OSCC submitted to elective neck dissection Variable Neck lymph-nodal status
Category pN+ (ONM) pN0
Overall survival (%) 5-year
10-year
50 67
29 58
P 0.030
ONM = occult lymph-node metastasis.
lower than those of RUSSOLO et al.31 (34%) and BYERS et al.7 (45%) in patients with OSCC. The immunostaining of VEGF-C in OSCC is a histopathological parameter that has been evaluated for others11,17,33,35,41 but generally few microscopic fields were analyzed and frequently the extension of the examined area and field magnification were not specified. The present study involved an objective and reproducible evaluation of VEGF-C expression in malignant cells of OSCC using a computer system that permitted capture of the tumor area to be analyzed by investigators. Thirty invasive front tumor fields were evaluated (X400 magnification) in each sample; without doubt, a representative area of the most invasive region of the OSCC. Use of this objective methodology minimizing the subjectivity and facilitates comparison of the results with those of future studies, an essential step for establishment of the real influence of VEGF-C expression by malignant cells on the clinical and biological behavior of the OSCC. According to the histopathological malignancy grading, most (79%) of the 87 OSCC cases were classified as well-tomoderately differentiated (more differentiated tumors), a finding consistent with other published studies11,35,41. There was no statistical correlation between malig-
nancy grading and VEGF-C expression (Table 2). Bryne’s grading system6 was used, in which the morphological features are determined from the most invasive front of the tumors. The invasive tumor front consists of the most aggressive cells, which have the ability to invade surrounding tissue structures, including vessels, and thereby metastasize. The characteristics of the invasive tumor front are of major significance for the prognosis of oral cancer3,5. VEGF-C expression in the malignant cells did not influence the likelihood of overall and disease-free survival for the patients with OSCC located in the tongue and floor of the mouth (Table 3). The only prognostic factor that reached statistical significance and influenced cumulative overall survival rates was the occurrence of occult lymph-node metastasis (pN+), as showed in Table 4 and Fig. 2. These results reinforce previous findings that, in the early stages of OSCC, the prediction of occult lymph-node metastasis is, at present, indispensable for improvement in the probability of survival1,7,10,11,12,31. The pursuit of predictive factors should be increased to reduce the morbidity of elective neck dissection in patients who do not have metastasis in the pathological exam (pN0). In conclusion, although VEGF-C expression has been occasionally regarded
Fig. 2. Cumulative overall survival probability curves for groups with lymph-node metastasis (pN+) and without lymph-node metastasis (pN0).
as a prognostic factor in OSCC, these results suggest that the isolated immunoexpression of VEGF-C in malignant cells does not directly influence the clinical outcome and prognosis of patients with early OSCC of tongue and floor of mouth. In spite of this, further studies should be performed with regard to VEGF-C, microvessel density and the occurrence of regional lymph-node metastases because, in the future, understanding the influence of lymphangiogenic growth factors and tumour immunomodulation may yield an array of new therapies to limit the metastatic spread of oral cancer. Acknowledgments. The authors thank FAPESP (Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo, grant #2005/ 04577-4) and CAPES (Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior) for supporting this study.
References 1. Amaral TMP, Freire ARS, Carvalho AL, Pinto CAL, Kowalski LP. Predictive factors of occult metastasis and prognosis of clinical stages I and II squamous cell carcinoma of the tongue and floor of the mouth. Oral Oncol 2004: 40: 780– 786. 2. Arinaga M, Noguchi T, Takeno S, Chujo M, Miura T, Uchida Y. Clinical significance of vascular endothelial growth factor C and vascular endothelial growth factor receptor 3 in patients with nonsmall cell lung carcinoma. Cancer 2003: 97: 457–464. 3. Ba`nkfalvi A, Piffko` J. Prognostic and predictive factors in oral cancer: the role of invasive tumor front. J Oral Path Med 2000: 29: 291–298. 4. Beasley NJP, Prevo R, Banerji S, Leek RD, Moore J, van Trappen P, Cox G, Harris AL, Jackson DG. Intratumoral lymphangiogenesis and lymph node metastasis in head and neck cancer. Cancer Res 2002: 62: 1315–1320. 5. Bryne M, Boysen M, Alfsen CG, Abeler VM, Sudbo J, Nesland JM, Kristensen GB, Piffko J, Bankfalvi A. The invasive front of carcinomas. The most important area for tumour prognosis? Anticancer Res 1998: 18: 4757–4764. 6. Bryne M, Koppang HS, Lilleng R, Stene T, Bang G, Dabelsteen E. New malignancy grading is a better prognostic indicator than Broders’ grading in oral squamous cell carcinoma. J Oral Pathol Med 1989: 18: 432–437. 7. Byers RM, El-Naggar AK, Lee YY, Rao B, Fornage B, Terry NH, Sample D, Hankins P, Smith TL, Wolf PJ. Can we detect or predict the presence of occult nodal metastases in patients with
VEGF-C expression and occult lymph-node metastasis in oral cancer
8.
9. 10.
11.
12.
13. 14.
15.
16.
17.
18.
19.
squamous carcinoma of the oral tongue? Head Neck 1998: 20: 138–144. Clarijs R, Ruiter DJ, De Wall RM. Lymphangiogenesis in malignant tumours: does it occur? J Pathol 2001: 193: 143–146. Ferrara N, Davis-Smyth T. The biology of vascular endothelial growth factor. Endocr Rev 1997: 18: 4–25. Haddadin KJ, Soutar DS, Oliver RJ, Webster MH, Robertson AG, MacDonald DG. Improved survival for patients with clinically T1/T2, N0 tongue tumors undergoing a prophylactic neck dissection. Head Neck 1999: 21: 517–525. Kishimoto K, Sasaki A, Yoshihama Y, Mese H. Expression of vascular endothelial growth factor-C predicts regional lymph node metastasis in early oral squamous cell carcinoma. Oral Oncol 2003: 39: 391–396. KOWALSKI LP, MAGRIN J, SAB_OIA M, SANTOS JC, TORLONI H. Squamous cell carcinoma of the tongue: a review of 629 patients treated at a single institution. South Am J Cancer 1997: 1: 245-254. Kowalski LP, Medina JE. Nodal metastases: predictive factors. Otolaryngol Clin North Am 1998: 31: 621–637. Krishnan J, Kirkin V, Steffen A, Hegen M, Weih D, Tomarev S, Wilting J, Sleeman JP. Differential in vivo and in vitro expression of vascular endothelial growth factor (VEGF)-C and VEGF-D in tumors and its relationship to lymphatic metastasis in immunocompetent rats. Cancer Res 2003: 63: 713–722. Kukk E, Lymboussaki. Taira S, Kaipanen A, Jeltsch M, Joukov V, Alitalo K. VEGF-C receptor binding, and pattern of expression with VEGFR-3 suggests a role in lymphatic vascular development. Development 1996: 122: 3829– 3837. Li QL, Chen FJ, Zeng ZY, Yang AK, Wu QL, Zhang HZ, Hou JH. Expression and clinical significance of VEGF-C and Flt-4 in tongue squamous cell carcinoma. Ai Zheng 2006: 25: 235–240. Lo´pez De Cicco R, Watson JC, Bassi DE, Litwin S, Klein-Szanto1 AJ. Simultaneous expression of furin and vascular endothelial growth factor in human oral tongue squamous cell carcinoma progression. Clin Cancer Res 2004: 10: 4480–4488. Munoz-Guerra MF, Marazuela EG, Martin-Villar E, Quintanilla M, Gamallo C. Prognostic significance of intratumoral lymphangiogenesis in squamous cell carcinoma of the oral cavity. A study of the early stages. Cancer 2004: 100: 553–560. Nakamura Y, Yasuoka H, Tsujimoto M, Yang Q, Tsukiyama A, Imabun S, Nakahara M, Nakao K, Nakamura M, Mori I, Kakudo K. Clinicopathological significance of vascular endothelial growth factor-C in breast carcinoma with
20.
21.
22.
23.
24.
25.
26.
27. 28.
29.
30.
31.
long-term follow-up. Mod Pathol 2003: 16: 309–314. Nakaya H, Kawashiri S, Tanaka A, Noguchi N, Kato K, Hase T, Yamamoto E. Influences of angiogenesis and lymphangiogenesis on cancerous invasion in experimentally induced tongue carcinoma. J Oral Pathol Med 2005: 34: 87–92. Nakazato T, Shingaki S, Kitamura N, Saito C, Kuwano R, Tachibana M. Expression level of vascular endothelial growth factor-C and -A in cultured human oral squamous cell carcinoma correlates respectively with lymphatic metastasis and angiogenesis when transplanted into nude mouse oral cavity. Oncol Rep 2006: 15: 825–830. Neuchrist C, Erovic BM, Handisurya A, Fischer MB, Steiner GE, Hollemann D, Gedlicka C, Saaristo A, Burian M. Vascular endothelial growth factor C and vascular endothelial growth factor receptor 3 expression in squamous cell carcinomas of the head and neck. Head Neck 2003: 25: 464–474. Nicosia RF. What is the role of vascular endothelial growth factor-related molecules in tumor angiogenesis? Am J Pathol 1998: 153: 11–16. O-Charoenrat P, Rhys-Evans P, Eccles SA. Expression of vascular endothelial growth factor family members in head and neck squamous cell carcinoma correlates with lymph node metastasis. Cancer 2001: 92: 556–568. Ohno F, Nakanishi H, Abe A, Seki Y, Kinoshita A, Hasegawa Y, Tatematsu M, Kurita K. Regional difference in intratumoral lymphangiogenesis of oral squamous cell carcinomas evaluated by immunohistochemistry using D240 and podoplanin antibody: an analysis in comparison with angiogenesis. J Oral Pathol Med 2007: 36: 281–289. Okamoto M, Nishimine M, Kishi M, Kirita T, Sugimura M, Nakamura M, Konishi N. Prediction of delayed neck metastasis in patients with stage I/ II squamous cell carcinoma of the tongue. J Oral Pathol Med 2002: 31(4):227–233. Pepper MS. Lymphangiogenesis and tumor metastasis: myth or reality? Clin Cancer Res 2001: 7: 462–468. Pepper MS, Tille JC, Nisato R, Skobe M. Lymphangiogenesis and tumor metastasis. Cell Tissue Res 2003: 314: 167– 177. Ribatti D, Nico B, Crivellato E, Roccaro AM, Vacca A. The history of the angiogenic switch concept. Leukemia 2007: 21: 44–52. Roskoski Jr R. Vascular endothelial growth factor (VEGF) signaling in tumor progression. Crit Rev Oncol Hematol 2007: 62: 179–213 Epub 2007 Feb 26.. Russolo M, Giacomarra V, Papanikolla L, Tirelli G. Prognostic indicators of occult metastases in oral cancer. Laryngoscope 2002: 112: 1320–1323.
377
32. Saharinen P, Tammela T, Karkkainen MJ, Alitalo K. Lymphatic vasculature: development, molecular regulation and role in tumor metastasis and inflammation. Trends Immunol 2004: 25: 387–395. 33. Sedivy R, Beck-Mannagetta J, Haverkampf C, Battistutti W, Ho¨nigschnabl S. Expression of vascular endothelial growth factor-C correlates with the lymphatic microvessel density and the nodal status in oral squamous cell cancer. J Oral Pathol Med 2003: 32: 455– 460. 34. Shayan R, Achen MG, Stacker SA. Lymphatic vessels in cancer metastasis: bridging the gaps. Carcinogenesis 2006: 27: 1729–1738. 35. Shintani S, Li C, Ishikawa T, Mihara M, Nakashiro K, Hamakawa H. Expression of vascular endothelial growth factor A, B, C and D in oral squamous cell carcinoma. Oral Oncol 2004: 40: 13–20. 36. Siriwardena BSMS, Kudo Y, Ogawa I, Udagama M.N.G.P.K.. Tilakaratne WM, Takata T. VEGF-C is associated with lymphatic status and invasion in oral cancer. J Clin Pathol 2007: 9 [Epub ahead of print].. 37. Skobe M, Hawighorst T, Jackson DG, Prevo R, Janes L, Velasco P, Riccardi L, Alitalo K, Claffey K, Detmar M. Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis. Nat Med 2001: 7: 192– 198. 38. Sobin LH, Fleming ID. TNM classification of malignant tumors, fifth edition. Union Internationale Contre le Cancer and the American Joint Committee on Cancer. Cancer 1997: 80: 1803–1804. 39. Sobin LH, Wittekind CH. TNM: classification of malignant tumors. 6th ed. New York: John Wiley & Sons 2002. 40. Soini Y, Puhakka A, Kahlos K, Sa¨ily M, Pa¨a¨kko¨ P, Koistinen P, Kinnula V. Endothelial nitric oxide synthase is strongly expressed in malignant mesothelioma but does not associate with vascular density or the expression of VEGF, Flk1 or Flt1. Histopathology 2001: 39: 179– 186. 41. Tanigaki Y, Nagashima Y, Kitamura Y, Matsuda H, Mikami Y, Tsukuda M. The expression of vascular endothelial growth factor –A and –C, and receptors 1 and 3: Correlation with lymph node metastasis and prognosis in tongue squamous cell carcinoma. Inter J Mol Med 2004: 14: 389–395. 42. Tsurusaki T, Kanda S, Sakai H, Kanetake H, Saito Y, Alitalo K, Koji T. Vascular endothelial growth factor-C expression in human prostatic carcinoma and its relationship to lymph node metastasis. Br J Cancer 1999: 80: 309– 313. 43. Veikkola T, Karkkainen M, Claesson-Welsh L, Alitalo K. Regulation
378
Faustino et al.
of angiogenesis via vascular endothelial growth factor receptors. Cancer Res 2000: 60: 203–212. 44. Warburton G, Nikitakis NG, Roberson P, Marinos NJ, Wu T, Sauk JJ, Ord RA, Wahl SM. Histopathological and lymphangiogenic parameters in relation to lymph node metastasis in early stage
oral squamous cell carcinoma. J Oral Maxilofac Surg 2007: 65: 475–484. Address: Denise Tostes Oliveira Faculdade de Odontologia de Bauru A´rea de Patologia Alameda Octa´vio Pinheiro Brisolla
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