Cervical lymph node metastasis is one of the

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1 ORIGINAL ARTICLE CORRELATION BETWEEN LYMPHATIC VESSEL DENSITY AND REGIONAL METASTASIS IN SQUAMOUS CELL CARCINOMA OF THE TONGUE Man Ki Chung, MD, 1 Jin-Young Min, MD, 1 Yoon Kyoung So, MD, 1 Young-Hyeh Ko, MD, PhD, 2 Han-Sin Jeong, MD, PhD, 1 Young-Ik Son, MD, PhD, 1 Chung-Hwan Baek, MD, PhD 1 1 Department of Otorhinolaryngology Head and Neck Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea. chbaek@skku.edu 2 Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea Accepted 28 May 2009 Published online 11 August 2009 in Wiley InterScience ( DOI: /hed Abstract: Background. The aim of this study was to investigate the correlation between lymphatic vessel density in squamous cell carcinoma of the tongue and regional metastasis. Methods. Intratumoral and peritumoral lymphatic densities (ILDs and PLDs, respectively) were determined by immunohistochemical staining of lymphatic endothelial cells with podoplanin in 62 patients surgically treated for tongue cancer. Clinicopathological variables were quantified, and their correlations with regional metastasis were assessed. Results. The rate of regional metastasis was significantly higher in patients with high ILD than that in those with low ILD (21/33, 63.6% vs 8/29, 27.5%; p ¼.006). Perineural invasion and lymphovascular invasion were also significantly correlated with regional metastasis. By multivariate analyses, ILD was the only variable identified to be significantly correlated with regional metastasis (p ¼.009). On the other hand, PLD showed no correlation with regional metastasis. Conclusions. ILD showed a strong correlation with regional metastasis in patients with squamous cell carcinoma of the Correspondence to: C.-H. Baek Contract grant sponsor: Samsung Biomedical Research Institute; contract grant number: SBRI C-A The first 2 authors contributed equally to this work. VC 2009 Wiley Periodicals, Inc. tongue. VC 2009 Wiley Periodicals, Inc. Head Neck 32: , 2010 Keywords: tongue; lymphatic vessel; squamous cell carcinoma; podoplanin; metastasis Cervical lymph node metastasis is one of the most important prognostic factors in oral tongue cancer. Many reports have tried to predict the presence of cervical metastasis using clinical and pathological variables, such as tumor thickness and depth of tumor invasion. 1,2 Because the lymphatic vessels provide important routes for the spread of solid tumors, the characteristics of lymphatic vessels and the process of lymphangiogenesis have received greater attention attributed to the recent introduction of specific lymphatic endothelial cell (LEC) markers, such as the homeobox-containing transcription factor Prox1, lymphatic vessel endothelial hyaluronan receptor (LYVE-1), and podoplanin. Among the many issues, 2 features that remain to be addressed with respect to the biological significance of lymphatic vessels are Lymphatic Vessel Density in Tongue Cancer HEAD & NECK DOI /hed April

2 whether the functioning vessels are new ones and whether they are intratumorally or peritumorally located. 3 In breast cancer, colorectal cancer, and melanoma, lymphatic vessel density has been shown to be correlated with clinical outcome, and it has even been proposed as a new target for antimetastatic treatment modalities. 4 6 However, few reports have been issued concerning the characteristics of lymphatic vessels in squamous cell carcinomas of the head and neck (HNSCCs). Furthermore, it is not clear whether intratumoral and peritumoral lymphatic vessel densities (ILDs and PLDs, respectively) are significantly correlated with regional metastasis or survival In fact, HNSCC has many different disease subgroups, depending on primary tumor sites and different oncologic behaviors. Therefore, the majority of previous studies on HNSCC have included different tumor subgroups, which makes the interpretation and clinical applications of these studies difficult. In the present study, we focused on squamous cell carcinoma of the tongue and quantified ILD and PLD in archived, surgically resected tongue cancer specimens by immunostaining LECs with monoclonal antibody against podoplanin (gp36). We then investigated the correlations between clinicopathological variables including ILD or PLD and the presence of cervical metastasis. MATERIALS AND METHODS Patients. The institutional review board of Samsung Medical Center (Association for the Accreditation of Human Research Protection Programs accredited) approved this retrospective analysis. The requirement for patient informed consent was waived because the study involved the use of pathological archives and retrospective clinical data. Patient private information was fully protected by coding. Sixty-two patients with squamous cell carcinoma in the oral tongue were enrolled. All patients had previously been observed with an untreated malignancy and underwent surgical treatment at Samsung Medical Center between 1996 and Surgical treatment included complete resection of primary tumor and elective or therapeutic neck dissection according to clinical N Table 1. Demographic data. Characteristic No. of patients (%) Age Range y Median 55 y Sex Male 37 (59.6) Female 25 (40.4) AJCC stage (pathologic) I 23 (37.2) II 11 (17.7) III 11 (17.7) IV 17 (27.4) Follow-up duration Range mo Median 43 mo Treatment failure rate 22/62 (35.4) Local 6 (9.6) Locoregional 4 (6.4) Regional 5 (8.0) Distant local, regional 7 (11.2) Abbreviation: AJCC, American Joint Committee on Cancer. classification status. Detailed patient characteristics are provided in Table 1. Twenty-three patients had pathologic stage I disease (37.2%), 11 patients had stage II or III (17.7%), and 17 patients (27.4%) had stage IV. Median duration of follow-up was 43 months (range, months). Treatment failure occurred in 22 of the 62 patients (35.4%). Local failure occurred in 6 patients (9.6%), locoregional in 4 patients (6.4%), regional in 5 patients (8.0%), and distant in 7 patients (11.2%). The 5-year disease-free and 5-year overall survival rates were 59.0% and 76.5%, respectively. Immunohistochemistry. A monoclonal antibody against human podoplanin (gp36; Acris Antibodies, Hiddenhausen, Germany) was used to stain LECs. A mouse monoclonal antibody to human CD34 type III (Chemicon International, Temecula, CA) was used to coimmunostain blood vessels. For staining, 4-lm sections were cut from paraffin blocks, deparaffinized in xylene, and hydrated in a series of graduated ethanol solutions (100%, 96%, 80%, 70%, and 50%). They were then heated in a microwave oven for 5 minutes in citrate buffer (ph 6.0) for antigen retrieval (repeated 2 times), and were washed for 5 minutes with Tris-buffered saline (TBS; ph 7.6) (repeated 3 times). Endogenous peroxidase activity was blocked with H 2 O 2 solution for 20 minutes. Primary antibody against podoplanin 446 Lymphatic Vessel Density in Tongue Cancer HEAD & NECK DOI /hed April 2010

3 (dilution, 1:50) was then applied for 18 hours, and primary antibody was applied to CD34 (dilution, 1:50) for 30 minutes. Sections were then washed 3 times with TBS, and secondary antibody (horseradish peroxidase linked) was applied for 20 minutes at room temperature. Streptavidin buffer was applied for 10 minutes, and 3,3 0 -diaminobenzidine was used as the chromogen. All sections were counterstained with hematoxylin and eosin. Tissue blocks from 10 tissue samples obtained from patients with normal oral mucosa were used as controls. Determination of Lymphatic Vessel Density. Examinations were performed by co-observation of 2 investigators (including a pathologist, Y.H.K.) who were blinded to clinical information. Quantitative assessment of lymphatic vessels by immunohistochemical staining of LECs was performed in sections that were stained for podoplanin, as described previously. 9 Lymphatic vessel density was defined as the number of lymphatic vessels per high-magnification field (100). At first, the entire section was scanned for the expression pattern of podoplanin, the tumor boundary, and the distribution of lymphatic vessels at low magnification (40). Three areas of tissue ( hot spot ) with distinctly increased numbers of lymphatic vessels were then selected from inside the tumor and areas within a distance of 500 lm from the tumor periphery, respectively. All immunostained vessels in each hot spot were counted at high magnification (100). As a control, lymphatic vessel density in normal oral mucosa was counted in the same manner. As a result, mean ILD, PLD, and value of lymphatic vessel density in normal mucosa were 16.2, 4.2, and 9.8, respectively. Statistical Analysis. Correlations between cervical lymph node metastasis and clinicopathological variables were analyzed using the Mann Whitney test, Pearson s chi-square test, and binary logistic regression. The clinicopathologic variables examined were age, sex, pathologic T and N classifications (according to the American Joint Committee on Cancer Staging System, sixth edition), histologic grade, PLD, ILD, perineural invasion, lymphovascular invasion, depth of tumor invasion, and the status of resection margin. ILD and PLD values were dichotomized using mean values as cutoffs. High ILD or high PLD was defined as ILD or PLD above each mean value, and low ILD or low PLD was defined as ILD or PLD at the same or below each mean value. For all tests, 2-tailed p values of <.05 were considered statistically significant. All analyses were performed using PASW RESULTS Podoplanin-positive lymphatic vessels were identified in all of the 62 tumor specimens examined. Lymphatic vessels had an irregular shape and a thin-walled structure. Subdermal lymphatic vessels were observed in the subepithelial portions of normal oral mucosa, and were clearly distinguished from adjacent blood vessels that were podoplain-negative and surrounded by smooth muscle. Intratumoral lymphatics generally presented slit-like shapes, and they were highly concentrated in hotspot areas, in which they were intermingled with and sometimes collapsed by pushing margins of tumor cell nests (Figures 1A and 1B). Peritumoral lymphatics were relatively sparse (Figure 1C). Tumor emboli were observed within the lymphatic vessel (Figure 1D). In comparisons between ILD and PLD in accord with clinicopathological variables, mean values of ILD were significantly higher than the mean values of PLD in any subgroup (Table 2). By univariate analysis, the rate of regional metastasis was significantly higher in patients with high ILD than that in patients with low ILD (21/33, 63.6% vs 8/29, 27.5%; p ¼.006) (Table 3). The presence of perineural invasion (present, 14/20, 70.0% vs absent, 15/42, 35.7%; p ¼.01) and lymphovascular invasion (present, 10/13, 76.9% vs absent, 19/49, 38.7%; p ¼.01) were also found to be significantly correlated with regional metastasis. By multivariate analysis, ILD was the only variable identified to be significantly correlated with regional metastasis (p ¼.009) (Table 3). However, PLD did not show any significant correlation with regional metastasis. The probability of cervical metastasis was investigated by linear discrimination analysis using combinations of variables that were found to be significant by univariate analysis. When high ILD, perineural invasion, and lymphovascular invasion were all present, the probability of cervical metastasis was 100%, and when high ILD and lymphovascular invasion were present, the probability was 88.8% (Table 4). Lymphatic Vessel Density in Tongue Cancer HEAD & NECK DOI /hed April

4 FIGURE 1. Immunohistochemical staining of lymphatic vessels with monoclonal antibody to podoplanin (gp36) in squamous cell carcinoma of the tongue. (A, B) Podoplanin-positive lymphatics (arrows) are interspersed between tumor nests (original magnifications, 100 and 200, respectively). They have narrow, slit-like lumens, and more often they are collapsed by pushing tumor margins. (C) Peritumoral lymphatics (arrow heads) are seen at the periphery of tumor margin (original magnification, 100). (D) Note the tumor embolus within the intratumoral lymphatic vessel (arrow) (original magnification, 400). DISCUSSION This study was undertaken to investigate the characteristics of lymphatic vessels in squamous cell carcinoma of the tongue. It was found that mean ILD values were significantly higher than mean PLD values in tongue cancer. Furthermore, a high ILD value was strongly correlated with regional metastasis by univariate and multivariate analyses. Reliable predictors of regional metastasis are of the utmost importance for planning treatment and predicting prognosis in HNSCC. In addition to changing the extent of surgical resection and the administration of postoperative adjuvant radiotherapy, the presence of regional metastasis directly affects survival and clinical outcomes. 1,11 15 Tumor lymphatics are attracting more research attention, given new evidence that they play an active role in lymphatic metastasis from primary tumors, and that they probably can supply new prognostic parameters to assess the risk of cervical metastasis in HNSCC. 16 Recent advances in lymphatic research followed the introduction of specific molecular markers for LECs, such as LYVE-1, Prox-1, D2-40, and gp36 for podoplanin. Yuan et al 17 reported podoplanin overexpression in tongue cancer and found this to be associated with a poor clinical outcome. In their study, D2-40 was used, which is a monoclonal antibody to podoplanin expressed in LECs and in certain tumor cells, such as squamous cell carcinoma. 18 They measured the podoplanin expression in lymphatic 448 Lymphatic Vessel Density in Tongue Cancer HEAD & NECK DOI /hed April 2010

5 Table 2. Comparisons between mean values of intratumoral and peritumoral lymphatic densities according to the clinicopathological variables. Variable No. of patients (%) Mean ILD Mean PLD p value* Age <55 y 29 (46.7) < y 33 (53.3) <.05 Sex Male 37 (59.6) <.05 Female 25 (40.4) <.05 Pathologic T classification T1 29 (46.7) <.05 T2 29 (46.7) <.05 p value <.05 N.S. Pathologic N classification N0 33 (53.3) <.05 Nþ 29 (46.7) <.05 p value <.05 N.S. Histologic differentiation Well 43 (69.3) <.05 Moderate/poor 19 (30.7) <.05 Extracapular lymph node spread Absent 51 (82.2) <.05 Present 11 (17.8) <.05 p value <.05 N.S. Lymphovascular invasion Absent 49 (79.1) <.05 Present 13 (20.9) <.05 Depth of tumor invasion 5 mm 19 (30.7) <.05 >5 mm 43 (69.3) <.05 Abbreviations: ILD, intratumoral lymphatic density; PLD, peritumoral lymphatic density; N.S., not significant. *p values between mean ILD and PLD, Mann Whitney test. p values within dichotomized subgroups, Mann Whitney test. T1 and T2 classifications were compared, excluding 4 patients with T3 or T4 classification. vessels and tumor cells together, whereas in the present study we used monoclonal antibody to podoplanin (gp36) and calculated lymphatic vessel density within tumors and at tumor peripheries. Nevertheless, our results concur with those of Yuan s study that ILD in tongue cancer correlates with regional metastasis. As mentioned earlier, HNSCC is composed of various disease subgroups that are differentiated by location, such as the oral tongue, the base of the tongue, the larynx, and hypopharynx, and each of these disease subgroups has different cervical metastasis rates and oncologic behavior. This diversity complicates direct comparisons of results regarding lymphangiogenesis in HNSCC. In a study by Kyzas et al, 9 the majority of tumors were located in the lower lip (62%), and the remainder were in the oral cavity or in the larynx (27% and 11%). The ILDs of lower lip tumors were significantly lower than those of other sites, although they still retained an association with the presence of lymph node metastasis at diagnosis. It is interesting that, although these tumors were squamous cell carcinomas, the characteristics of lymphangiogenesis differed according to tumor site. Squamous cell carcinomas of different anatomic origins may vary in terms of their lymphangiogenic properties and be influenced by organ- and region-specific factors. In a study on the complex relationships between ILD and neck node metastasis by disease subgroup, Beasley et al 7 suggested that different HNSCC subgroups, although equally lymphangiogenic, may have substantially different capacities in terms of invading the lymphatic system. Lymphatic Vessel Density in Tongue Cancer HEAD & NECK DOI /hed April

6 Table 3. Correlation between regional metastasis and clinicopathological variables. pn0 (n ¼ 33) pnþ (n ¼ 29) Variable No. (%) No. (%) Univariate p value* Multivariate p value Age <55 y 15 (45.4) 14 (48.2) y 18 (54.5) 15 (51.7) Sex Male 20 (60.6) 17 (58.6).87 Female 13 (39.3) 12 (41.3) Pathologic T classification T1 T2 32 (96.9) 26 (89.6).73 T3 T4 1 (3.0) 3 (10.3) Histologic differentiation Well 24 (72.7) 19 (65.6) Moderate/poor 9 (2.2) 10 (34.4) PLD Low 15 (45.4) 18 (62.0) High 18 (54.5) 11 (37.9) ILD Low 21 (63.6) 8 (27.5) High 12 (36.3) 21 (72.4) Perineural invasion Absent 27 (81.8) 15 (51.7) Present 6 (18.1) 14 (48.2) Lymphovascular invasion Absent 30 (90.0) 19 (65.5) Present 3 (9.0) 10 (34.4) Depth of tumor invasion 5 mm 13 (39.3) 6 (20.6) >5 mm 20 (60.6) 23 (79.3) Resection margin 5 mm 18 (54.5) 15 (51.7).88 <5 mm 15 (45.4) 14 (48.2) Abbreviations: PLD, peritumoral lymphatic density; ILD, intratumoral lymphatic density. *Pearson s chi-square test. Binary logistic regression test. Peritumoral lymphatic density, dichotomized by mean value. Intratumoral lymphatic density, dichotomized by mean value. The main advantages of our study are that we used lymphatic vessel density, which is a widely accepted methodology, 7 9 rather than intratumoral lymphatic vessel positivity and negativity. In addition, we analyzed the lymphatic characteristics of the oral tongue cancer only, which makes clinical implication of this study straightforward. One issue that remains to be resolved is the exact role of ILD and PLD in the spread of tumors. In breast cancer, the down-regulation of LYVE-1 in intratumoral lymphatics suggests that it has little clinical significance; in HNSCC, however, debate continues concerning the roles of lymphatic vessels, although in tongue cancer it appears that ILD is greater than that of PLD and that ILD can predict the presence of cervical metastasis, according to recent reports. 7,16 In the present study, we found that ILD, but not PLD, is elevated in squamous cell carcinoma of the tongue. This result concurs with previous reports, in which ILD was found to be elevated more in Table 4. Probability for regional metastasis in accord with combinations of variables. Combination of variables* No. (probability, %) High ILD þ PNI þ þ LVI þ 4/4 (100.0) High ILD þ LVI þ 8/9 (88.8) PNI þ þ LVI þ 5/6 (83.3) High ILD þ PNI þ 10/13 (76.9) PNI þ LVI 9/32 (28.1) Low ILD þ LVI 5/22 (22.7) Low ILD þ PNI 4/20 (20.1) Low ILD þ PNI þ LVI 3/18 (16.6) Abbreviations: ILD, intratumoral lymphatic density; PNI þ, perineural tumor invasion present; PNI, perineural tumor invasion absent; LVI þ, lymphovascular tumor invasion present; LVI, lymphovascular tumor invasion absent. *High/low ILD: dichotomized by mean value. 450 Lymphatic Vessel Density in Tongue Cancer HEAD & NECK DOI /hed April 2010

7 tongue cancer that in any other HNSCC subgroups. 9,10 This finding implies that lymphatic vessels play an active role in the oncologic behavior of tongue cancer, and offers a direction to those involved in target-therapy research. The main limitations of the present study are short follow-ups of patients and the lack of evidence concerning whether the lymphatic vessels stained by podoplanin were newly formed ones or preexisting ones, which could have been resolved by double staining with Ki-67 proliferating marker. Furthermore, this study does not provide a biological rationale that explains the correlation between ILD and regional metastasis. In summary, the present study demonstrates that ILD is more predominant than PLD, and that ILD is strongly correlated with regional metastasis in squamous cell carcinoma of the tongue. REFERENCES 1. Asakage T, Yokose T, Mukai K, et al. Tumor thickness predicts cervical metastasis in patients with stage I/II carcinoma of the tongue. Cancer 1998;82: Rasgon BM, Cruz RM, Hilsinger RL Jr, Sawicki JE. Relation of lymph-node metastasis to histopathologic appearance in oral cavity and oropharyngeal carcinoma: a case series and literature review. Laryngoscope 1989;99: Ji RC. Lymphatic endothelial cells, tumor lymphangiogenesis and metastasis: new insights into intratumoral and peritumoral lymphatics. Cancer Metastasis Rev 2006;25: Schoppmann SF, Bayer G, Aumayr K, et al. Prognostic value of lymphangiogenesis and lymphovascular invasion in invasive breast cancer. Ann Surg 2004;240: Straume O, Jackson DG, Akslen LA. Independent prognostic impact of lymphatic vessel density and presence of low-grade lymphangiogenesis in cutaneous melanoma. Clin Cancer Res 2003;9: Matsumoto K, Nakayama Y, Inoue Y, et al. Lymphatic microvessel density is an independent prognostic factor in colorectal cancer. Dis Colon Rectum 2007;50: Beasley NJ, Prevo R, Banerji S, et al. Intratumoral lymphangiogenesis and lymph node metastasis in head and neck cancer. Cancer Res 2002;62: Franchi A, Gallo O, Massi D, Baroni G, Santucci M. Tumor lymphangiogenesis in head and neck squamous cell carcinoma: a morphometric study with clinical correlations. Cancer 2004;101: Kyzas PA, Geleff S, Batistatou A, Agnantis NJ, Stefanou D. Evidence for lymphangiogenesis and its prognostic implications in head and neck squamous cell carcinoma. J Pathol 2005;206: 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. Cancer 2004;100: Shintani S, Matsuura H, Hasegawa Y, Nakayama B, Fujimoto Y. The relationship of shape of tumor invasion to depth of invasion and cervical lymph node metastasis in squamous cell carcinoma of the tongue. Oncology 1997;54: Sparano A, Weinstein G, Chalian A, Yodul M, Weber R. Multivariate predictors of occult neck metastasis in early oral tongue cancer. Otolaryngol Head Neck Surg 2004;131: Spiro RH, Guillamondegui O Jr, Paulino AF, Huvos AG. Pattern of invasion and margin assessment in patients with oral tongue cancer. Head Neck 1999;21: Thompson SH. Cervical lymph node metastases of oral carcinoma related to the depth of invasion of the primary lesion. J Surg Oncol 1986;31: Woolgar JA, Scott J. Prediction of cervical lymph node metastasis in squamous cell carcinoma of the tongue/ floor of mouth. Head Neck 1995;17: Maula SM, Luukkaa M, Grenman R, Jackson D, Jalkanen S, Ristamaki R. Intratumoral lymphatics are essential for the metastatic spread and prognosis in squamous cell carcinomas of the head and neck region. Cancer Res 2003;63: Yuan P, Temam S, El-Naggar A, et al. Overexpression of podoplanin in oral cancer and its association with poor clinical outcome. Cancer 2006;107: Schacht V, Dadras SS, Johnson LA, Jackson DG, Hong YK, Detmar M. Up-regulation of the lymphatic marker podoplanin, a mucin-type transmembrane glycoprotein, in human squamous cell carcinomas and germ cell tumors. Am J Pathol 2005;166: Lymphatic Vessel Density in Tongue Cancer HEAD & NECK DOI /hed April