Clinicopathologic significance of CD105-assessed microvessel density in glottic laryngeal squamous cell carcinoma

Auris Nasus Larynx 37 (2010) 77–83 www.elsevier.com/locate/anl

Clinicopathologic significance of CD105-assessed microvessel density in glottic laryngeal squamous cell carcinoma Elvir Zvrko a,*, Anton Mikic b, Ljiljana Vuckovic c a

b

Clinic for Otorhinolaryngology and Maxillofacial Surgery, Clinical Center of Montenegro, Podgorica, Montenegro Institute for Otorhinolaryngology and Maxillofacial Surgery, School of Medicine, University of Belgrade, Belgrade, Serbia c Center for Pathology and Forensic Medicine, Clinical Center of Montenegro, Podgorica, Montenegro Received 28 December 2008; accepted 20 May 2009 Available online 23 June 2009

Abstract Objective: Intratumoral microvessel density (MVD) determined with the use of antibodies to endoglin (CD105) is considered to be an important prognostic marker in a variety of malignancies. The purpose of this study has been to analyze the clinicopathologic significance of CD105-assessed MVD in SCCs primary localized in glottic region of larynx. Methods: Surgical specimens from 40 patients with resected glottic squamous cell carcinomas were immunostained for CD105. CD105assessed MVD was calculated at 400 magnification. Using the mean MVD as a cut-off, tumors were classified in the ‘‘high MVD’’ group and in the ‘‘low MVD’’ group. Clinicopathologic data were collected retrospectively. Results: The mean MVD assessed by CD105 in considered glottic SCCs was 12.3 (standard deviation [SD] = 3.65). MVD varied among tissue samples from 5 to 21 (median 12.5). High MVD was significantly correlated with a more aggressive tumor phenotype, including T3–T4 tumor (Fisher exact test, P = 0.004) and advanced clinical stage (Fisher exact test P = 0.026). Kruskal–Wallis test identified significant relation between pT stages and CD105-assessed MVD (P = 0.011). CD105-assessed MVD was significantly related to malignancy recurrence presence/absence (Mann–Whitney U-test P = 0.023). Logistic regression in multivariate modality showed that MVD (odds ratio [OR] 2.29, P = 0.033, 95% confidence interval [CI] 1.06–7.53) and advanced T category (T3–T4) (OR 4.11, P = 0.026, 95% CI 2.38–9.46) were significantly related to malignancy recurrence presence/absence. Cox regression analysis revealed that expression of CD105 (P = 0.031) and N status (P = 0.014) were the independent factors for disease-free survival. Conclusion: High expression of CD105 correlated significantly with advanced T status and locoregional recurrence. The present preliminary results suggest that CD105-assessed MVD in primary glottic squamous cell carcinomas may identify patients at risk of disease recurrence. # 2009 Elsevier Ireland Ltd. All rights reserved. Keywords: Angiogenesis; CD105; Mean vascular density; Laryngeal carcinoma; Prognosis

1. Introduction Angiogenesis, or neo-vascularization, is the term used for the growth and development of new capillary vessels. Tumor angiogenesis, the formation of peritumor and intratumor new blood vessels, is necessary to the growth and metastasis of solid tumors by supplying nutrient and oxygen, disposing metabolites and releasing growth factors that promote tumor cell proliferation, tumor progression in the host stroma and * Corresponding author at: Ljubljanska bb, Clinical Center of Montenegro, 81000 Podgorica, Montenegro. Tel.: +382 69 401 431. E-mail address: [email protected] (E. Zvrko).

metastasis development [1]. Solid tumors cannot grow beyond 1–2 mm in diameter without angiogenesis [2]. Angiogenesis is a complex multistep process involving extracellular matrix remodeling, endothelial cell migration and proliferation, microvessel differentiation and anastomosis. These processes are tightly controlled by positive and negative angiogenic factors and their receptors that regulate one or more of these key events [3,4]. However, the regulatory mechanisms in angiogenesis are not yet fully understood. Intratumoral microvessel density (MVD) is considered a promising prognostic marker in a variety of malignancies and increased MVD correlated with malignancy progression

0385-8146/$ – see front matter # 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.anl.2009.05.005

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and shorter overall and relapse-free survival rates. Intratumoral MVD has been previously studied using panendothelial markers such as CD34, CD31 and Factor VIII, which cannot distinguish proliferating endothelium in tissue undergoing angiogenesis from normal pre-existing blood vessels. In contrast to panendothelial antibodies, endoglin (CD105) antibodies have shown a greater specificity for tumor vasculature. Endoglin is a 180-kDa homodimeric type-I transmembrane glycoprotein that modulates transforming growth factor-b (TGF-b) signaling by interacting with TGF-b receptors I and III [5]. The gene encoding for CD105 is approximately 40 kb long and it has been mapped to human chromosome 9q34 [6]. Li et al. [7] demonstrated that hypoxia activates the CD105 gene promoter, augmenting its mRNA transcription and protein translation. Several studies have defined the role of CD105 as a powerful marker to quantify MVD in solid and hematopoietic tumors including breast cancer [8,9], cutaneous melanoma [10], colon cancer [11–13], esophageal cancer [14], lung cancer [15,16], gynecological malignancies [17,18], renal cancer [19], head and neck cancer [20–24] and in multiple myeloma [25] and hairy cell leukemia [26]. To the best of our knowledge, only a few studies investigated endoglin expression in laryngeal squamous cell carcinomas (SCCs). The purpose of the present study has been to analyze for the first time the clinicopathologic significance of MVD in SCCs primary localized only in glottic region of larynx using anti-CD105 monoclonal antibody.

2. Materials and methods 2.1. Patients Medical charts of patients who had been treated for primary laryngeal SCCs at the Clinic for Otorhinolaryngology and Maxillofacial Surgery of the Clinical center of Montenegro in Podgorica from 2003 to 2008 were reviewed. The clinical information, including sex, age, histologic and nuclear grade, primary tumor (T) classification, nodal (N) status, TNM stage, and oncological outcome, were obtained retrospectively from clinical records. In the analysis of clinical data we have defined poor oncological outcome as recurrence of disease or occurrence of metastasis after treatment. Forty cases of laryngeal invasive SCCs localized in glottic region with available follow-up data were evaluated, 20 of them had early cancer (stage I or II) and 20 had advanced cancer (stage III or IV). All selected patients underwent complete resection as primary treatment. Patients with second primaries or who had received primary radiotherapy and/or chemotherapy were not considered. Treatment decision-making was based on clinical stage and on the presence or not of lymph node metastasis at the time of diagnosis. Partial laryngectomy was performed in 22 and

Table 1 Clinicopathologic characteristics of 40 patients with glottic squamous cell carcinoma. No. of patients

(%)

Sex Male Female

29 11

(72.5) (27.5)

T stage T1 T2 T3 T4

12 10 16 2

(30) (25) (40) (5)

N stage N0 N1 N2

36 3 1

(90) (7.5) (2.5)

TNM stage I II III IV

12 8 18 2

(30) (20) (45) (5)

Histological grading G1 G2

23 17

(57.5) (42.5)

Nuclear grading G1 G2 G3

14 24 2

(35) (60) (5)

total laryngectomy in 18 patients. Nine patients received neck dissection operation simultaneously to the primary tumor removal and lymph node metastasis was presented in four cases. Eight patients underwent postoperative radiotherapy. Mean follow-up time (calculated in months from treatment completion to the last otolaryngological control) was 20.5 months (range 6–60 months). Pathological staging was determined according to the 6th TNM Classification of Malignant Tumors of the International Union Against Cancer. Clinicopathologic characteristics of the selected patients are shown in Table 1. 2.2. Immunohistochemistry Forty tissue blocks of glottic SCCs were fixed in 10% formalin and embedded in paraffin wax. All included samples originated from complete resection material. We selected the best section from each block showing central and peripheral areas of the tumor, avoiding areas with necrosis. One 4-mm-thick section from each block was cut and deparaffinized. The sections were pretreated with Proteinase K for 5 min. The endogenous peroxides’ activity was blocked with H2O2 solution in methanol for 10 min. The slides were incubated with mouse monoclonal antibody CD105 (clone SN6h diluted 1:20, DAKO, Denmark) and washed with TRIS-buffered saline. Diaminobezidine (DAB) was used as a chromogen. The slides were then counterstained with hematoxylin. Laryngeal tissues free of tumor

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Fig. 1. High endoglin expression in laryngeal squamous cell carcinoma. Activated endothelia are marked with brown staining (original magnification 200).

Fig. 2. Low endoglin expression in laryngeal squamous cell carcinoma (original magnification 200).

were used as positive controls, and the primary antibody was replaced with TRIS-buffered saline for negative controls.

determine the effect of distinct prognostic factors on disease-free survival, a multivariate analysis was performed according to the Cox regression model. A P value less than 0.05 was considered to be significant in all statistical analyses. All statistical analyses were conducted with SPSS (Statistical Package for Social Sciences, SPSS Inc., Chicago, IL, USA).

2.3. Quantification of microvessel density The intratumoral MVD quantification was performed under light microscopy in accordance with the procedure reported by Weidner et al. [27] The sections were scanned at 40 magnification to select the four areas with the highest vascular density (‘‘hot spots’’). CD105-positive endothelial cells were counted using a 400 magnification. The rounded mean value of the vessel count in four fields for each case was used as the final MVD value. For statistical analyses, the mean MVD value of all cases was used as the cut-off point to separate tumors with high vs. low microvessel density, as previously described [28]. Any endoglin-stained single cell or cell cluster that was clearly separated from the adjacent microvessels, tumor cells or other elements of connective tissue was considered to be a countable vessel. A visible vascular lumen was not required to count as a microvessel. All counts were performed by a pathologist (Lj. V.) who did not know the clinical data. Fig. 1 shows a representative field of invasive laryngeal carcinoma with high CD105 expression. For comparison, Fig. 2 depicts a representative field of carcinoma with less staining for CD105.

3. Results The studied population consisted of 40 patients with glottic SCCs, from which 29 were males and 11 females, with a mean age of 63.2 years (range 44–81 years). The mean MVD value assessed by CD105 in considered glottic SCCs was 12.3 (standard deviation [SD] = 3.65). MVD varied among tissue samples from 5 to 21 (median 12.5). The distribution of microvessel counts among 40 patients with laryngeal cancer is shown in Fig. 3. Using the mean MVD as a cut-off, 20 (50%) tumors were classified in the

2.4. Statistical analysis The correlation between the clinicopathologic parameters and the expressions of CD105 were evaluated using the Fisher exact test, Mann–Whitney U-test and Kruskal– Wallis test. Logistic regression model in multivariate modality for binary outcomes was also applied to evaluate the association between oncologic outcome after treatment, MVD and other pathological features. The disease-free survival rate was analyzed using Kaplan–Meier method, and statistical significance was assessed by the log-rank test. To

Fig. 3. The distribution of microvessel counts among 40 patients with glottic cancer.

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Table 2 The correlation of CD105-assessed MVD with clinicopathologic parameters in patients with glottic squamous cell carcinoma. Variables

CD105 expressiona

Number of patients

Low

High

Sex Male Female

29 11

16 4

13 7

Age (years) 65 >65

23 17

9 11

14 6

T classification T1 and T2 T3 and T4

22 18

16 4

6 14

N status N0 N+

36 4

18 2

18 2

NSb

NS

.004

NS

TNM stage I and II III and IV

20 20

14 6

6 14

Histologic grade G1 G2 and G3

23 17

8 12

15 5

Nuclear grade G1 G2 and G3

14 26

6 14

8 12

a b

P value

.026

NS

NS

Low microvessel density (MVD) below 12.3, high MVD above 12.3. Not significant.

‘‘high MVD’’ group and the rest, 20 (50%) tumors, consisted the ‘‘low MVD’’ group. The correlation of CD105-assessed MVD with clinicopathologic parameters is summarized in Table 2. High MVD was significantly correlated with a more aggressive tumor phenotype, including T3–T4 tumor (Fisher exact test P = 0.004) and advanced clinical stage (Fisher exact test P = 0.026). There was no significant correlation between the expression of CD105 and age, sex, histological differentiation grade or nuclear grade. Also, Fisher exact test did not show any statistically significant difference in glottic SCCs MVD between pN+ and pN0/cN0 malignancies. Mean CD105-assessed MVD was 11.2 (SD 2.98) in pT1 carcinomas, 10.2 (SD 2.39) in pT2 carcinomas, 14.37 (SD 3.99) in pT3, 13 (SD 0) in pT4. We performed a nonparametric test for trend across order groups (modified Kruskal–Wallis test) to identify differences in MVD between pT stages and identified significant differences (P = 0.011). Mean CD105-assessed MVD was 11.2 (SD 2.98) in stage I carcinomas, 10.37 (SD 2.56) in stage II, 13.83 (SD 4.1) in stage III and 13 (SD 0) in stage IV carcinomas. Kruskal–Wallis test showed statistical trend for relation between stage grouping and CD105-assessed MVD (P value = 0.062). Eight of 40 patients with laryngeal SCCs developed locoregional recurrence (3 local recurrences, 5 recurrences to neck lymph nodes; mean CD105-assessed MVD 15.25;

Fig. 4. Box-plot graph of the distribution of MVD in the groups with and without locoregional recurrence. The box corresponds to the interquartile ranges, with the lower boundary of the box representing the 25th percentile and the upper boundary representing the 75th percentile. Median MVD values are indicated with the line inside the box. The vertical line represents minimal and maximal value.

median 15; SD 4.5). In the group without locoregional recurrence mean MVD was 11.6 (median 12; SD 3.1). CD105-assessed MVD was significantly related to malignancy recurrence presence/absence (Mann–Whitney U-test P = 0.023) (Fig. 4). Logistic regression in multivariate modality considering age, gender, histological grade, tumor extension, lymph node metastases, clinical stage and CD105-assessed MVD showed that MVD (odds ratio [OR] 2.29, P = 0.033, 95% confidence interval [CI] 1.06–7.53) and advanced T category (T3–T4) (OR 4.11, P = 0.026, 95% CI 2.38– 9.46) were significantly related to malignancy recurrence presence/absence. Advanced T (T3 and T4) category (logrank P = 0.036), the presence of lymph node metastasis at the time of diagnosis (log-rank P = 0.029), and high CD105assessed MVD (log-rank P = 0.047) were associated with worse disease-free survival. Furthermore, Cox regression analysis revealed that expression of CD105 (P = 0.031) and N status (P = 0.014) were the independent factors for disease-free survival.

4. Discussion There is growing evidence supporting MVD as an important predictor of tumor behavior in a number of human malignancies. However, several studies have failed to find that MVD is a significant prognostic factor. These discrepancies may reflect the differences in the endothelial markers used for immunohistochemical staining. Previous studies used panendothelial markers, such as CD31, CD34, and von Willebrand factor (Factor VIII), to evaluate MVD. Transmembrane glucoprotein CD31 stains both large and small vessels with equal intensity, as well as blood vessels in

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normal and tumor tissue, and occasionally stains carcinoma cells [29]. CD34 can express on endothelial cells, especially those undergoing active angiogenesis, but specificity is compromised since it can also stain a variety of other mesenchymal cells [30]. Emoto et al. [31] found that it was difficult to identify native or newborn vessels by using CD34. Von Willebrand factor stains large vessels with high sensitivity and capillaries with variable and focal staining [32] and can also stain lymphatics [33]. Therefore the use of antibodies specifically reacting with endothelial cells of angiogenic tissue reduces the probability of false-positive staining. Immunohistochemical studies using anti-CD105 antibodies have shown that anti-CD105 reacts with newborn vessels in neoplastic tissues. Recently, several studies have examined endoglin expression in solid and hematopoietic tumors including breast [8,9], prostate [34], esophageal [14], cervical [35], colorectal [11,12], non-small cell lung cancer cancers [15,16], and found endoglin as a more specific and sensitive microvessel marker than other commonly used panendothelial antibodies. Only few studies considered endoglin expression to determine MVD in head and neck SCCs. Kyzas et al. [24] investigated MVD determined with CD105 in 108 patients with head and neck SCC. High MVD was associated with a more aggressive tumor phenotype, including advanced clinical stage and the presence of lymph node metastasis at the time of diagnosis. Also, high MVD was an independent predictor of mortality in the subgroup of patients with squamous cell carcinoma of the oral cavity and larynx. In 2002, Schimming and Marme [36] showed that endoglin expression was significantly higher in SCCs of the oral cavity than in adjacent normal mucosa, in SCCs of the oral cavity with histologically proven regional metastases than in cases without metastases and by T2, T3 and T4 SCCs of the oral cavity than by T1 tumors. Ma˘rga˘ritescu et al. [37] did not observe any significant association of CD105 expression with age, sex, primary tumor’s location, clinical stage or differentiation grade in oral squamous cell carcinoma but they concluded that CD105 expression is up-regulated in oral squamous cell carcinoma and has a significant role in the development of such malignancies. Marioni et al. [38] investigated the relation between CD105-assessed MVD and pathological features among 13 cases of oral and oropharyngeal SCCs with lymph node metastases (pN+) and 13 pN0 cases. MVD was significantly higher in the group of patients with locoregional malignancy recurrence than in the group without recurrence. Multivariate logistic regression that considered MVD, pT and pN showed that CD105-assessed MVD was the only parameter significantly related with patients’ post-treatment outcome. In 2006, Chuang et al. [39] concluded that the expression of CD105 is a useful predictive prognostic factor in early tongue cancer because the higher expression of CD105 in the tumor bed implicates a more aggressive potential for T1 and T2 tongue cancers. Chien et al. [20] studied CD105 expression in 73 cases of hypopharyngeal SCC. The high expression of

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CD105 correlated with positive N metastasis and advanced TNM stage (stage IV vs. stage II and III). By use of univariate analysis, patients with high expression of CD105 in carcinoma tissue showed the trend toward a worse 5-year survival rate. In 2005, Marioni et al. [21] evaluated CD105 expression in nine head and neck basaloid squamous cell carcinomas site-and stage-matched with nine cases with SCC. CD105-assessed mean area fractions occupied by vessels were not significantly different between the two groups of malignancies. CD105 expression in laryngeal cancer was investigated only in few studies. Martone et al. [22] studied CD105 expression among 127 consecutive cases of head and neck primary SCCs and 107 of them were laryngeal malignancies. CD105-assessed MVD was significantly higher in N+ carcinomas. The patients with high MVD had a significantly shorter disease-free and overall survival. Multivariate analysis showed that a high MVD was the only independent marker of carcinoma recurrence or death. Marioni et al. [40] investigated the relation between immunohistochemically CD105-assessed MVD and pathological (pT, pN stage, and G) and prognostic features (recurrence of diseases) in laryngeal SCC. Thirteen out of the 43 considered cases of laryngeal SCCs developed malignant locoregional recurrence after treatment and multivariate logistic regression confirmed that CD105-assessed MVD was significantly related to disease recurrence presence/absence. In 2008, Marioni et al. [41] concluded that CD105 expression correlated with carcinoma recurrence after treatment and shorter disease-free interval and suggest that CD105 expression may be useful to detect cervical node-negative patients with a higher risk of early laryngeal carcinoma recurrence. In this study, we analyzed for the first time the clinicopathologic significance of CD105 expression among patients with laryngeal squamous cell carcinomas primary localized in glottic region. Because of known differences between glottic and supraglottic tumors we wanted to have a homogenous group of patients. CD105 microvessel staining was consistently present in all the cases studied. This is in agreement with previous studies where endoglin reacted mainly with proliferating blood vessels [34]. Our study found that MVD marked with CD105 antibody was significantly associated with a more aggressive tumor phenotype, including T3–T4 tumor (P = 0.004) and advanced clinical stage (P = 0.026), a finding that is in line with those of previous studies [20]. There was no significant correlation between the expression of CD105 and age, sex, differentiation grade or nuclear grade. Also, Fisher exact test did not show any statistically significant difference in glottic SCCs MVD between pN+ and pN0/cN0 malignancies. The lack of statistical association between MVD and N0/N+ category was possibly due to the limited size of our preliminary series. The studies by Chien et al. [20], Martone et al. [22], Kyzas et al. [24], and Schimming and Marme [34] concluded that CD105-assessed MVD was significantly

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higher in N+ head and neck SCCs. Marioni et al. [40] did not find significantly higher CD105 expression in N+ laryngeal cancer. We found significant relation between T stage grouping and CD105-assessed MVD (P = 0.011) and statistical trend for relation between TNM stage grouping and CD105 expression (P = 0.062) what is not confirmed in study of Marioni et al. [40]. Also, we investigated CD105-assessed MVD role in predicting patients’ prognosis and analyzed correlation between CD105-assessed MVD and development of locoregional recurrence. We had eight patients who developed locoregional recurrence (3 local recurrences and 5 recurrences to neck lymph nodes) and mean MVD was 15.25. In the group without locoregional recurrence mean MVD was 11.6. Statistical analysis showed a significant difference between CD105-assessed MVD in poor (with locoregional recurrence) and good outcome groups (Mann– Whitney U-test = 0.023). Logistic regression in multivariate modality showed that MVD (P = 0.033) and advanced T category (T3–T4) (P = 0.026) were significantly related to malignancy recurrence presence/absence. Martone et al. [22], Marioni et al. [40], and Marioni et al. [41] also found that CD105-assessed MVD was significantly related to disease recurrence. By use of univariate analysis (the log-rank test) in our study, patients with high expression of CD105 (P = 0.047) in tumor tissue, advanced T (T3 and T4) category (P = 0.036) and the presence of lymph node metastasis at the time of diagnosis (P = 0.029) showed the worse disease-free survival rate. Besides N status, CD105 expression also showed the prognostic value for survival in multivariate analysis according to the Cox regression model (P = 0.031). Our observations support findings of Marioni et al. [41] for the prognostic significance of CD105 in laryngeal SCCs.

5. Conclusion MVD has the potential to be used as a prognostic marker. A marker that is strictly specific for tumor vasculature probably does not exist, but CD105 antibodies have shown a greater specificity for tumor vasculature in comparison with panendothelial markers. Our data showed that high expression of CD105 in microvessels of primary glottic laryngeal SCCs correlated significantly with advanced T status and locoregional recurrence. Results of the present study suggest that CD105-assessed MVD may be useful to identify patients with more aggressive disease who are at risk of recurrence of disease and developing regional metastasis. The prognostic significance of MVD determined with CD105 requires verification in larger studies of tumor from the same anatomic location and treated with the same therapeutic modality before it can be incorporated into clinical practice.

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