Vascular endothelial growth factors C and D and lymphangiogenesis at the early stage of esophageal squamous cell carcinoma progression

Vascular endothelial growth factors C and D and lymphangiogenesis at the early stage of... SUMMARY We conducted a detailed study of lymphangiogenesis and subsequent lymph node metastasis in early-stage esophageal squamous cell carcinoma (ESCC) using immunostaining for D2-40 and vascular endothelial growth factor (VEGF)-C and D. The study materials included 13 samples of normal squamous epithelium, 6 samples of low-grade intraepithelial neoplasia (LGIN), and 60 samples of superficial ESCC (M1 and M2 cancer 24; M3 or deeper cancer 36). We assessed lymphatic vessel density (LVD) using D2-40 and immunoreactivity for VEGF-C and D in relation to histological type, lymphatic invasion, and lymph node metastasis. LVD in M1 and M2 lesions and M3 or deeper lesions was significantly higher than in normal squamous epithelium (P < 0.001). High expression of VEGF-C and D was observed in M1 and M2 cancer and in M3 or deeper cancer, but not in normal squamous epithelium or LGIN. LVD in VEGF-C- and D-positive cases was significantly higher than in negative cases (P < 0.001). In M3 or deeper cancer, the correlation between VEGF-C or D status and lymphatic invasion or lymph node metastasis was not significant. LVD in cases with positive lymphatic invasion and those with lymph node metastasis was significantly higher than in cases lacking either (P = 0.02 and 0.03, respectively). ESCC cells produce VEGF-C and D from the very early stage of progression. VEGF-C and D activate lymphangiogenesis, and this increase of lymphatic vessels leads to lymphatic invasion and subsequent lymph node metastasis. ABBREVIATIONS M1 carcinoma in situ M2 tumor invasion to the lamina propria mucosae M3 tumor invasion to the muscularis mucosa SM1 tumor invasion to the upper third of the submucosal layer SM2 tumor invasion to the middle third of the submucosal layer SM3 tumor invasion to the lower third of the submucosal layer The depth of invasion of superficial esophageal carcinoma is expressed in accordance with the subclassification criteria of the Japan Esophageal Society (Guidelines for clinical and pathologic studies on carcinoma of the esophagus).1 INTRODUCTION Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies worldwide.2 The prognosis of patients with ESCC is considered to be relatively poor because of the high risk of metastasis even at the early stage of carcinogenesis.3 The presence of lymph node metastasis is one of the most important factors determining the prognosis of ESCC.4 Lymph node metastasis of early-stage ESCC is present in approximately 0% of M1 or M2 cancer, 10% of M3 or SM1 cancer, and 50% of SM2 or SM3 cancer.5 However, little is known about the mechanism of lymph node metastasis or lymphovascular invasion at the early stage of cancer progression. Before the advent of a reliable marker of lymphatic vessels, lymphatic invasion had been diagnosed using hematoxylin and eosin (H&E) staining, which reveals cancer cells floating within the endothelial-lined space. However, lymphatic invasion cannot be recognized as complete occlusion of lymphatic vessels by cancer cells because the endothelial cells are too thin to be discerned easily, and the cancer cells are recognized as cancer nests. The lymphatic endothelium is reported to show strong expression of the marker antigen D2-40,6 and many studies have reported the usefulness of immunostaining with anti-D2-40 monoclonal antibody for identification of lymphatic invasion in the cancer stroma or even cancer nests of ESCC.7–11 It has been reported that vascular endothelial growth factor C (VEGF-C) and D (VEGF-D) induce not only angiogenesis but also lymphangiogenesis.12–15 In addition, VEGF-C and VEGF-D expression is reported to be significantly correlated with lymphovascular density (LVD), lymphatic invasion, and lymph node metastasis in advanced ESCC.16–25 However, details of lymphangiogenesis at the early stage of ESCC remain unclear. In this study, we focused on lymphangiogenesis at the early stage of ESCC progression in relation to lymph node metastasis using immunostaining for D2-40, VEGF-C, and VEGF-D. MATERIALS AND METHODS Tissue samples For this study, we employed 79 samples from 59 patients who had undergone histological examinations at Saitama Medical Center, Saitama Medical University, between 2006 and 2014. The tissue samples comprised 13 specimens of normal squamous epithelium, 6 specimens of low-grade intraepithelial neoplasia (LGIN), and 60 specimens of superficial ESCC (M1: 12 lesions, M2: 12 lesions, M3: 10 lesions, SM1: 8 lesions, SM2: 4 lesions, SM3: 14 lesions). The tissue samples were obtained by esophageal biopsy (n = 3), endoscopic resection (n = 23), or esophagectomy with lymph node dissection (n = 53). The 13 samples of normal squamous epithelium were all located distantly from the cancer lesion in esophagectomy specimens. We excluded specimens from patients who had undergone radiotherapy and/or chemotherapy before lesion resection. For analysis of lymph node metastasis, we evaluated cases showing M3 or deeper cancer invasion only. We excluded five cases in which further advanced cancers coexisted and eight cases for which endoscopic resection had been performed for analysis of lymph node metastasis. Pathological diagnosis was made according to the Guidelines for Clinical and Pathologic Studies on Carcinoma of the Esophagus (10th ed.).26 Sections were cut from 3-mm-wide step-sectioned blocks of endoscopic resection specimens, or from 5-mm-wide blocks obtained from surgically resected esophagi, and stained with H&E. We divided the specimens into four different histological types, i.e. normal squamous epithelium, LGIN, M1-M2 cancer, and M3 or deeper cancer, by reference to the frequency of lymph node metastasis. The study was performed under a protocol approved by our hospital ethics committee. Immunohistochemical staining Tissue samples fixed in 10% formalin and embedded in paraffin were cut into sections 4 μm thick and mounted on slides. Immunohistochemical staining of D2-40 was performed using the streptavidin–biotin–peroxidase method. The slides were immunostained with mouse anti-D2-40 monoclonal antibody (clone D2-40 (Nichirei Bioscience Co., Tokyo, Japan); ready to use) using a Ventana Bentimark XT machine (Ventana, Tuscon, Arizona, USA). Immunostaining for VEGF-C and VEGF-D was performed with a Leica Bond-III automatic immunostainer (Leica Biosystems Newcastle Ltd., UK). The slides were immunostained with mouse anti-VEGF-C monoclonal antibody (clone F-10 (Santa Cruz Biotechnology, Texas, USA) 1:1000) and mouse anti-VEGF-D monoclonal antibody (clone #78,923 (R &D Systems, Minneapolis, USA); 1:100). Counter staining was performed using hematoxylin (Hematoxylin 3G (Sakura Finetek Japan, Tokyo, Japan)). Quantification of lymphatic vessel density On the basis of D2-40 immunostaining, LVD in the lamina propria mucosae was assessed in normal esophageal mucosa, LGIN, and M1 and M2 cancer. For M3 and submucosal cancer, we counted the lymphatic vessels at the periphery and in the center of the tumor. Sections were screened according to the method described by Weidner et al.27 Briefly, using low magnifying power, three areas of strongest D2-40 staining (hot spots) were noted. Vessel counting was performed in a ×200 field, and the mean count for three fields was determined as the LVD. Evidence of a visible lymphatic vessel lumen was not required. Quantification of VEGF-C and D Expression of VEGF-C and D in over 50% of the cells examined was considered positive. Expression of VEGF-C and D was evaluated in each of three fields in D2-40 hot spots at ×200 magnification using a light microscope. All assessments were performed by two independent investigators (YK and TT) who were blinded to the clinical data. Statistical analysis Data are expressed as median and range. Correlations between the groups were assessed by chi-squared test. Differences between groups were analyzed by Mann-Whitney U test and Kruskal-Wallis test followed by Dunn's test. Differences at P < 0.05 were considered significant. RESULTS Expression of D2-40, VEGF-C and D in esophageal lesions Immunostaining for each of D2-40, VEGF-C and D in normal squamous epithelium, M1 cancer, M2 cancer, and submucosal cancer is shown in Figure 1. D2-40 expression was detected in lymphatic endothelial cells. In the normal esophageal mucosa, lymphatic vessels were found mainly in the lamina propria mucosae. Basal layer cells in some specimens of normal epithelium showed positivity for D2-40 (Fig. 1a). In lesions of M1 and M2 cancer, hot spots of D2-40-positive lymphatic vessels were found in the lamina propria mucosae (Fig. 1b,c). In lesions of M3 or deeper cancer, hot spots were located at the periphery of the tumor, in the cancer stroma, and inside cancer nests (Fig. 1d). Fig. 1 View largeDownload slide Expression of D2-40, VEGF-C, and VEGF-D in normal squamous epithelium and esophageal cancer (counterstaining with hematoxylin). (a–d) Immunohistochemical staining for D2-40 (×100). (e–h) Immunohistochemical staining for VEGF-C (×100). i-l: Immunohistochemical staining for VEGF-D (×100). (a): Normal squamous epithelium stained for D2-40. Lymphatic vessels are visible as brown capillaries. Lymphatic vessels are frequently found in the lamina proproia mucosae rather than the submucosal layer. Basal layer cells in some cases are positive for D2-40. (b,c): M1 (b) and M2 (c) cancer lesions stained for D2-40. Lymphatic vessels are massively aggregated in the lamina propria mucosae. (d): Submucosal cancer stained for D2-40. Lymphatic invasion (arrows) can be seen in the cancer stroma. (e, i): Normal squamous epithelium stained for VEGF-C (e) and VEGF-D (i). Both VEGF-C and D are negative in normal squamous epithelium, but are weakly positive in the duct of an esophageal gland proper (arrows). (f, g): Positive immunoreactivity for VEGF-C in case of M1 (f) and M2 (g) cancer, and for VEGF-D in cases of M1 (j) and M2 (k) cancer (×100). Cancerous lesions are clearly demarcated from the normal squamous epithelium (f). (h, l): VEGF-C (h) and D (l) in submucosal cancer (×100). Homogeneously stained cancer nests can be seen. Mononuclear cells that have infiltrated the cancer stroma are also positive for VEGF-C and D. Fig. 1 View largeDownload slide Expression of D2-40, VEGF-C, and VEGF-D in normal squamous epithelium and esophageal cancer (counterstaining with hematoxylin). (a–d) Immunohistochemical staining for D2-40 (×100). (e–h) Immunohistochemical staining for VEGF-C (×100). i-l: Immunohistochemical staining for VEGF-D (×100). (a): Normal squamous epithelium stained for D2-40. Lymphatic vessels are visible as brown capillaries. Lymphatic vessels are frequently found in the lamina proproia mucosae rather than the submucosal layer. Basal layer cells in some cases are positive for D2-40. (b,c): M1 (b) and M2 (c) cancer lesions stained for D2-40. Lymphatic vessels are massively aggregated in the lamina propria mucosae. (d): Submucosal cancer stained for D2-40. Lymphatic invasion (arrows) can be seen in the cancer stroma. (e, i): Normal squamous epithelium stained for VEGF-C (e) and VEGF-D (i). Both VEGF-C and D are negative in normal squamous epithelium, but are weakly positive in the duct of an esophageal gland proper (arrows). (f, g): Positive immunoreactivity for VEGF-C in case of M1 (f) and M2 (g) cancer, and for VEGF-D in cases of M1 (j) and M2 (k) cancer (×100). Cancerous lesions are clearly demarcated from the normal squamous epithelium (f). (h, l): VEGF-C (h) and D (l) in submucosal cancer (×100). Homogeneously stained cancer nests can be seen. Mononuclear cells that have infiltrated the cancer stroma are also positive for VEGF-C and D. Normal squamous cells exhibited no or only faint staining for both VEGF-C and D. Ductal cells of esophageal glands proper in normal esophageal tissue showed positive immunoreactivity for VEGF-C and D (Fig. 1e,i). VEGF-C and D expression was evident in the cytoplasm of the cancer cells. In M1 and M2 cancer, expression of both VEGF-C and D was clearly demarcated from the adjacent normal squamous epithelium (Fig. 1f,g,j,k). In cases of M3 or deeper cancer, positivity was even, and no heterogeneity was observed in cancer nests (Fig. 1h,l). In stromal tissue, some mononuclear cells also exhibited positivity for VEGF-C and D. Correlation of histological type with lymphatic vessel density and expression of VEGF-C and D (Table 1) Table 1 Correlation between each histological type and LVD, VEGF-C and D Lymphatic vessel density† VEGF-C‡ VEGF-D‡ (Median (range)) Negative (%) Positive (%) Negative (%) Positive (%) Normal (n = 13) 7.3 (3.0–10.3) 13 (100) 0 (0) 13 (100) 0 (0) LGIN (n = 6) 11.2 (6.5–18.0) 6 (100) 0 (0) 6 (100) 0 (0) M1 and M2 (n = 24) 13.5 (5.3–34.3)§ 9 (37.5) 15 (62.5) 13 (54.2) 11 (45.8) M3 or deeper (n = 36) 14.8 (7.3–27.0)§ 11 (30.6) 25 (69.4) 20 (55.6) 16 (44.4) Lymphatic vessel density† VEGF-C‡ VEGF-D‡ (Median (range)) Negative (%) Positive (%) Negative (%) Positive (%) Normal (n = 13) 7.3 (3.0–10.3) 13 (100) 0 (0) 13 (100) 0 (0) LGIN (n = 6) 11.2 (6.5–18.0) 6 (100) 0 (0) 6 (100) 0 (0) M1 and M2 (n = 24) 13.5 (5.3–34.3)§ 9 (37.5) 15 (62.5) 13 (54.2) 11 (45.8) M3 or deeper (n = 36) 14.8 (7.3–27.0)§ 11 (30.6) 25 (69.4) 20 (55.6) 16 (44.4) †Kruskal-Wallis test P < 0.001; ‡Chi-square test P < 0.005; §Dunn test P < 0.01. LGIN, low-grade intraepithelial neoplasia; LVD, lymphatic vessel density; VEGF, vascular endothelial growth factor. View Large Table 1 Correlation between each histological type and LVD, VEGF-C and D Lymphatic vessel density† VEGF-C‡ VEGF-D‡ (Median (range)) Negative (%) Positive (%) Negative (%) Positive (%) Normal (n = 13) 7.3 (3.0–10.3) 13 (100) 0 (0) 13 (100) 0 (0) LGIN (n = 6) 11.2 (6.5–18.0) 6 (100) 0 (0) 6 (100) 0 (0) M1 and M2 (n = 24) 13.5 (5.3–34.3)§ 9 (37.5) 15 (62.5) 13 (54.2) 11 (45.8) M3 or deeper (n = 36) 14.8 (7.3–27.0)§ 11 (30.6) 25 (69.4) 20 (55.6) 16 (44.4) Lymphatic vessel density† VEGF-C‡ VEGF-D‡ (Median (range)) Negative (%) Positive (%) Negative (%) Positive (%) Normal (n = 13) 7.3 (3.0–10.3) 13 (100) 0 (0) 13 (100) 0 (0) LGIN (n = 6) 11.2 (6.5–18.0) 6 (100) 0 (0) 6 (100) 0 (0) M1 and M2 (n = 24) 13.5 (5.3–34.3)§ 9 (37.5) 15 (62.5) 13 (54.2) 11 (45.8) M3 or deeper (n = 36) 14.8 (7.3–27.0)§ 11 (30.6) 25 (69.4) 20 (55.6) 16 (44.4) †Kruskal-Wallis test P < 0.001; ‡Chi-square test P < 0.005; §Dunn test P < 0.01. LGIN, low-grade intraepithelial neoplasia; LVD, lymphatic vessel density; VEGF, vascular endothelial growth factor. View Large The median LVD (range) revealed by D2-40 staining in the normal esophageal mucosa, LGIN, M1-M2 cancer, and M3 or deeper cancer was 7.3 (3.0–10.3), 11.2 (6.5–18.0), 13.5 (5.3–34.3), and 14.8 (7.3–27.0), respectively. The median LVD based on D2-40 immunostaining was lowest in the normal esophageal mucosa, followed in ascending order by LGIN, M1-M2 cancer, and M3 or deeper cancer, and the difference was significant by Kruskal-Wallis test (P < 0.001). Post hoc Dunn's test revealed that the LVD for M1-M2 cancer and M3 or deeper cancer was significantly higher than that for normal squamous epithelium (P < 0.001). Expression of both VEGF-C and D was not considered positive in normal squamous epithelium and LGIN. In LGIN, weak expression of VEGF-C and D was found in 4/6 and 2/6 lesions, respectively, but less than 50% of cells were positive. High expression of VEGF-C and D was observed in M1 and M2 cancer (62.5% and 45.8%, respectively) and in M3 or deeper cancer (69.4% and 44.4%, respectively). Chi-squared test showed that the expression of VEGF-C and D was significantly correlated between the groups (P < 0.005). Correlation between LVD and expression of VEGF-C or D in ESCC Figure 2 shows the correlation between LVD and the expression of VEGF-C or D in ESCC. The median (range) LVD was 15.2 (5.3–34.3) in VEGF-C-positive cases and 11.3 (6.3–23.0) in VEGF-C-negative cases, the difference being significant (P < 0.001). Similarly, the median (range) LVD was 17.0 (9.0–34.3) in VEGF-D-positive cases and 13.0 (5.3–23.0) in VEGF-D-negative cases, the difference also being significant (P < 0.001). Fig. 2 View largeDownload slide Lymphatic vessel density in relation to positivity or negativity for VEGF-C and D. (a) VEGF-C (median (range)). Negative: 11.3 (6.3–23.0), Positive: 15.2 (5.3–34.3), Mann-Whitney U test P < 0.005. (b) VEGF-D (median (range)). Negative: 13.0 (5.3–23.0), Positive: 17.0 (9.0–34.3), Mann-Whitney U test P < 0.005. Fig. 2 View largeDownload slide Lymphatic vessel density in relation to positivity or negativity for VEGF-C and D. (a) VEGF-C (median (range)). Negative: 11.3 (6.3–23.0), Positive: 15.2 (5.3–34.3), Mann-Whitney U test P < 0.005. (b) VEGF-D (median (range)). Negative: 13.0 (5.3–23.0), Positive: 17.0 (9.0–34.3), Mann-Whitney U test P < 0.005. Correlation between expression of VEGF-C and D, LVD, and lymphatic invasion or lymph node metastasis (Table 2) Table 2 Correlation between lymphatic invasion or lymph node metastasis and VEGF-C, D, and LVD VEGF-C VEGF-D Lymphatic vessel density Negative Positive Negative Positive (median (range)) Lymphatic invasion  Negative 9 14 15 8 13.7 (7.3–27.0)  Positive 2 11 5 8 18.7 (9.0–25.7)† P = 0.14 P = 0.12 Lymph node metastasis  Negative 3 8 5 6 13.0 (7. 7–25.3)  Positive 3 9 4 8 18.2 (13.0–27.0)† P = 0.90 P = 0.55 VEGF-C VEGF-D Lymphatic vessel density Negative Positive Negative Positive (median (range)) Lymphatic invasion  Negative 9 14 15 8 13.7 (7.3–27.0)  Positive 2 11 5 8 18.7 (9.0–25.7)† P = 0.14 P = 0.12 Lymph node metastasis  Negative 3 8 5 6 13.0 (7. 7–25.3)  Positive 3 9 4 8 18.2 (13.0–27.0)† P = 0.90 P = 0.55 †Mann-Whitney U test P < 0.05. View Large Table 2 Correlation between lymphatic invasion or lymph node metastasis and VEGF-C, D, and LVD VEGF-C VEGF-D Lymphatic vessel density Negative Positive Negative Positive (median (range)) Lymphatic invasion  Negative 9 14 15 8 13.7 (7.3–27.0)  Positive 2 11 5 8 18.7 (9.0–25.7)† P = 0.14 P = 0.12 Lymph node metastasis  Negative 3 8 5 6 13.0 (7. 7–25.3)  Positive 3 9 4 8 18.2 (13.0–27.0)† P = 0.90 P = 0.55 VEGF-C VEGF-D Lymphatic vessel density Negative Positive Negative Positive (median (range)) Lymphatic invasion  Negative 9 14 15 8 13.7 (7.3–27.0)  Positive 2 11 5 8 18.7 (9.0–25.7)† P = 0.14 P = 0.12 Lymph node metastasis  Negative 3 8 5 6 13.0 (7. 7–25.3)  Positive 3 9 4 8 18.2 (13.0–27.0)† P = 0.90 P = 0.55 †Mann-Whitney U test P < 0.05. View Large Lymphatic invasion was found in 13 of 36 M3 or deeper lesions (36.1%; M3: 0/10, SM1:3/8, SM2:1/4, SM3:9/14), whereas no lymphatic invasion was found in M1 and M2 lesions. Analysis of the M3 or deeper group revealed no correlation between the expression of VEGF-C or D and lymphatic invasion (P = 0.14 and 0.12, respectively). However, the median (range) LVD in cases positive for lymphatic invasion (18.7 (9.0–25.7)) was significantly higher than in cases that were negative (13.7 (7.3–27.0)) (P = 0.02). Lymph node metastasis was found in 12 of 23 cases of surgically resected M3 or deeper cancer (52.2%; M3: 1/3, SM1:0/4, SM2:1/3, SM3:10/13). We also found no correlation between VEGF-C or D expression and lymph node metastasis (P = 0.90 and 0.55, respectively). The median (range) LVD in cases that were positive for lymph node metastasis (18.2 (13.0–27.0)) was significantly higher than in cases that were negative (13.0 (9.0–25.3)) (P = 0.03). DISCUSSION Understanding the changes that occur in lymphatic vessels during cancer progression would undoubtedly help to clarify the mechanisms of lymph node metastasis and carcinogenesis. Accurate knowledge of the dynamic changes at the early stage of cancer progression might provide clues to chemoprevention, molecular targeting therapy, or early detection of cancer. Lymphatic invasion is well known to be a useful marker for prediction of lymph node metastasis.7,9–11 D2-40 has been shown to be a selective marker of the lymphatic endothelium and facilitates the objective identification of lymphatic invasion in comparison with conventional HE staining.10 In addition, D2-40 immunostaining makes it possible to assess the degree of lymphangiogenesis based on the LVD count. It has been reported that LVD is associated with lymph node metastasis and prognosis of gastric cancer, SCC of the uterine cervix, and head and neck SCC.28–30 The use of D2-40 immunostaining has shown that 0–5% of cases of M1 and M2 ESCC are positive for lymphatic invasion, whereas 35–60% of cases of M3 or deeper cancer are positive.5,10 These results are in agreement with our present findings. Similarly, some previous studies found no lymph node metastasis in cases of M1 and M2 ESCC, whereas 25–40% of M3 or deeper cases were positive.5,9,10 Our finding that 52.2% of cases of M3 or deeper ESCC were positive for lymph node metastasis suggested a higher incidence than that reported previously.5,9,10 One of the reasons for this high incidence of lymph node metastasis may have been that we excluded eight cases of M3 or deeper cancer treated using endoscopic mucosal resection. None of these cases treated by endoscopic resection showed clinical evidence of lymph node metastasis or distant metastasis at least two years after treatment. If these cases had been counted as lymph node metastasis negative, the incidence of lymph node metastasis would have been 38.7%. VEGF, especially VEGF-A, is known as to be a major inducer of angiogenesis and vessel permeability.31 Five additional members of the VEGF family—VEGF-B, VEGF-C, VEGF-D, VEGF-E, and placenta growth factor (PIGF)—have been characterized.32 Among them, VEGF-C and D are known to induce both angiogenesis and lymphangiogenesis.13,15 These cytokines act as ligands for vascular endothelial growth factor receptor (VEGFR)-2, which is expressed on vascular endothelial cells and is essential for formation of blood vessels.33 In addition, both cytokines can bind to VEGFR-3, which is expressed predominantly in the endothelium of lymphatic vessels.34 Thus, VEGF-C and D are reported to be the main inducers of lymphangiogenesis in various malignant tumors, including ESCC.14,16–25 The immunohistochemical positivity rate for VEGF-C is reported to be 40–100% in ESCC.14–24 With regard to VEGF-D in ESCC, two articles have reported positivity rates of 73.1% and 65.9%.24,25 Only one article has reported the positivity rate for both VEGF-C and D in ESCC. In that report, the VEGF-C positivity rate in cases of esophageal neoplasia (including esophageal dysplasia) was higher than the positivity rate for VEGF-D.24 This finding is similar to that in our present report. However, these previous studies14–25 included only a limited number of superficial ESCC cases, and thus the VEGF-C and D status at the early stage of ESCC progression was unclear. In this study, we examined 60 lesions of superficial ESCC and 6 lesions of LGIN. We divided the cancer lesions into two groups (i.e. M1 and M2 cases vs. M3 or deeper cases) in accordance with the reported rate of lymph node metastasis. The positivity rates for VEGF-C and D in M1 and M2 cases were 62.5% and 45.8%, respectively, being similar to the rate in M3 or deeper cases. On the other hand, all of the specimens of normal squamous epithelium and LGIN were judged as negative for both VEGF-C and D. In addition, LVD in M1 and M2 cases was double that for normal squamous epithelium, and the difference between the two groups was significant. LVD in M3 or deeper cases was slightly higher than that in M1 and M2 cases, and the difference was not significant. In addition, cases of ESCC positive for both VEGF-C and D showed a significantly higher LVD (P < 0.001) than cases that were negative, suggesting that both VEGF-C and D may play important roles in the recruitment of lymphatic ducts, and that lymphangiogenesis is activated at the very early stage of carcinogenesis. VEGFR-3, as well as VEGF-C, reveals significant correlation with LVD in ESCC.16 Overexpression of the VEGF-C may induce tumor lymphangiogenesis by stimulating VEGFR-3 expression on lymphatic endothelial cells. These findings will be a hint of further study. It has been reported that expression of VEGF-C and D is significantly correlated with lymphatic invasion, lymph node metastasis, and prognosis in various types of cancer including ESCC.16–25 Omoto et al.16 investigated lymphangiogenesis of ESCC (both superficial and advanced) in detail using immunohistochemistry for VEGF-C, VEGFR-3, and D2-40, and found that VEGF-C positivity was significantly correlated with lymphatic invasion, lymph node metastasis, and LVD. On the other hand, Noguchi et al. reported that VEGF-C expression showed no correlation with lymphatic invasion, lymph node metastasis, and prognosis. This study revealed that LVD was significantly higher in M3 or deeper cancer lesions with lymphatic invasion and lymph node metastasis than in those negative for lymphatic invasion and lymph node metastasis. However, no significant correlation was found between the expression of VEGF-C or D and lymphatic invasion or lymph node metastasis. In addition, despite the high rate of VEGF-C and D positivity and high level of LVD in M1-M2 cancers (similar to M3 or deeper cancers), none of the cases showed lymphatic invasion or lymph node metastasis. Therefore, other factors associated with induction of lymphatic invasion and subsequent lymph node metastasis may exist. Overall, the present findings suggest that cancer cells produce VEGF-C and D from the very early stage of ESCC progression. VEGF-C and D activate lymphangiogenic switching, but may not induce lymphatic invasion and lymph node metastasis directly. These data suggest that the increase in the number of lymphatic vessels leads to lymphatic invasion and subsequent lymph node metastasis at the early stage of ESCC progression. Acknowledgment This study was supported by MEXT KAKENHI Grant number 26461047. We are grateful to Tomoyuki Kawada for his kind advice and confirmation of statistical analysis. Human rights statement and informed consent All procedures followed were in accordance with the ethical standards of the committees responsible for human experimentation (institutional and national) and with the Helsinki Declaration of 1964 and later versions. Informed consent for inclusion of tissue samples in this study was obtained from all patients or their representatives. Notes All of the authors who are listed in the title page contributed significantly to this paper, and match the authorship that is stated in the “Guidelines of the international committee of Medical Journal Editors.” Conflict of interest disclosure: Youichi Kumagai received a research grant from MEXT KAKENHI. References 1 The Japan Esophageal Society . Japanese classification of esophageal cancer, tenth edition: part I . Esophagus 2009 ; 6 : 1 – 25 . CrossRef Search ADS 2 Enzinger P C , Mayer R J . Esophageal cancer . N Engl J Med 2003 ; 349 : 2241 – 52 . Review . 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Intratumoral lymphangiogenesis of esophageal squamous cell carcinoma and relationship with regulatory factors and prognosis . Pathol Int 2008 ; 58 : 611 – 9 . Google Scholar CrossRef Search ADS PubMed 19 Noguchi T , Takeno S , Shibata T , Uchida Y , Yokoyama S , Müller W . VEGF-C expression correlates with histological differentiation and metastasis in squamous cell carcinoma of the esophagus . Oncol Rep 2002 ; 9 : 995 – 9 . Google Scholar PubMed 20 Matsumoto M , Natsugoe S , Okumura H et al. Overexpression of vascular endothelial growth factor-C correlates with lymph node micrometastasis in submucosal esophageal cancer . J Gastrointest Surg 2006 ; 10 : 1016 – 22 . Google Scholar CrossRef Search ADS PubMed 21 Kimura Y , Watanabe M , Ohga T et al. Vascular endothelial growth factor C expression correlates with lymphatic involvement and poor prognosis in patients with esophageal squamous cell carcinoma . Oncol Rep 2003 ; 10 : 1747 – 51 . Google Scholar PubMed 22 Kitadai Y , Amioka T , Haruma K et al. Clinicopathological significance of vascular endothelial growth factor (VEGF)-C in human esophageal squamous cell carcinomas . Int J Cancer 2001 ; 93 : 662 – 6 . Google Scholar CrossRef Search ADS PubMed 23 Okazawa T , Yoshida T , Shirai Y et al. Expression of vascular endothelial growth factor C is a prognostic indicator in esophageal cancer . Hepatogastroenterology 2008 ; 55 : 1503 – 8 . Google Scholar PubMed 24 Ishikawa M , Kitayama J , Kazama S , Nagawa H . The expression pattern of vascular endothelial growth factor C and D in human esophageal normal mucosa, dysplasia and neoplasia . Hepatogastroenterology 2004 ; 51 : 1319 – 22 . Google Scholar PubMed 25 Tzao C , Lee S C , Tung H J et al. Expression of hypoxia-inducible factor (HIF)-1alpha and vascular endothelial growth factor (VEGF)-D as outcome predictors in resected esophageal squamous cell carcinoma . Dis Markers 2008 ; 25 : 141 – 8 . Google Scholar CrossRef Search ADS PubMed 26 The Japan Esophageal Society . Japanese classification of esophageal cancer, tenth edition: parts II and III . Esophagus 2009 ; 6 : 71 – 94 . CrossRef Search ADS 27 Weidner N , Semple J P , Welch W R , Folkman J . Tumor angiogenesis and metastasis: correlation in invasive breast carcinoma . N Engl J Med 1991 ; 324 : 1 – 8 . Google Scholar CrossRef Search ADS PubMed 28 Wang X L , Fang J P , Tang R Y , Chen X M . Different significance between intratumoral and peritumoral lymphatic vessel density in gastric cancer: a retrospective study of 123 cases . BMC Cancer 2010 ; 10 : 299 . Google Scholar CrossRef Search ADS PubMed 29 Gombos Z , Xu X , Chu C S , Zhang P J , Acs G . Peritumoral lymphatic vessel density and vascular endothelial growth factor C expression in early-stage squamous cell carcinoma of the uterine cervix . Clin Cancer Res 2005 ; 11 : 8364 – 71 . Google Scholar CrossRef Search ADS PubMed 30 Maula S M , Luukkaa M , Grénman R , Jackson D , Jalkanen S , Ristamäki 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 : 1920 – 6 . Google Scholar PubMed 31 Leung D W , Cachianes G , Kuang W J , Goeddel D V , Ferrara N . Vascular endothelial growth factor is a secreted angiogenic mitogen . Science 1989 ; 246 : 1306 – 9 . Google Scholar CrossRef Search ADS PubMed 32 Veikkola T , Alitalo K . VEGFs, receptors and angiogenesis . Semin Cancer Biol 1999 ; 9 : 211 – 20 . Review . Google Scholar CrossRef Search ADS PubMed 33 Veikkola T , Karkkainen M , Claesson-Welsh L , Alitalo K . Regulation of angiogenesis via vascular endothelial growth factor receptors . Cancer Res 2000 ; 60 : 203 – 12 . Google Scholar PubMed 34 Kaipainen A , Korhonen J , Mustonen T et al. Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during development . Proc Natl Acad Sci 1995 ; 92 : 3566 – 70 . Google Scholar CrossRef Search ADS PubMed © The Authors 2018. Published by Oxford University Press on behalf of International Society for Diseases of the Esophagus. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Diseases of the Esophagus Oxford University Press

Vascular endothelial growth factors C and D and lymphangiogenesis at the early stage of esophageal squamous cell carcinoma progression

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The International Society for Diseases of the Esophagus
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© The Authors 2018. Published by Oxford University Press on behalf of International Society for Diseases of the Esophagus.
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1120-8694
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1442-2050
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10.1093/dote/doy011
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Abstract

SUMMARY We conducted a detailed study of lymphangiogenesis and subsequent lymph node metastasis in early-stage esophageal squamous cell carcinoma (ESCC) using immunostaining for D2-40 and vascular endothelial growth factor (VEGF)-C and D. The study materials included 13 samples of normal squamous epithelium, 6 samples of low-grade intraepithelial neoplasia (LGIN), and 60 samples of superficial ESCC (M1 and M2 cancer 24; M3 or deeper cancer 36). We assessed lymphatic vessel density (LVD) using D2-40 and immunoreactivity for VEGF-C and D in relation to histological type, lymphatic invasion, and lymph node metastasis. LVD in M1 and M2 lesions and M3 or deeper lesions was significantly higher than in normal squamous epithelium (P < 0.001). High expression of VEGF-C and D was observed in M1 and M2 cancer and in M3 or deeper cancer, but not in normal squamous epithelium or LGIN. LVD in VEGF-C- and D-positive cases was significantly higher than in negative cases (P < 0.001). In M3 or deeper cancer, the correlation between VEGF-C or D status and lymphatic invasion or lymph node metastasis was not significant. LVD in cases with positive lymphatic invasion and those with lymph node metastasis was significantly higher than in cases lacking either (P = 0.02 and 0.03, respectively). ESCC cells produce VEGF-C and D from the very early stage of progression. VEGF-C and D activate lymphangiogenesis, and this increase of lymphatic vessels leads to lymphatic invasion and subsequent lymph node metastasis. ABBREVIATIONS M1 carcinoma in situ M2 tumor invasion to the lamina propria mucosae M3 tumor invasion to the muscularis mucosa SM1 tumor invasion to the upper third of the submucosal layer SM2 tumor invasion to the middle third of the submucosal layer SM3 tumor invasion to the lower third of the submucosal layer The depth of invasion of superficial esophageal carcinoma is expressed in accordance with the subclassification criteria of the Japan Esophageal Society (Guidelines for clinical and pathologic studies on carcinoma of the esophagus).1 INTRODUCTION Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies worldwide.2 The prognosis of patients with ESCC is considered to be relatively poor because of the high risk of metastasis even at the early stage of carcinogenesis.3 The presence of lymph node metastasis is one of the most important factors determining the prognosis of ESCC.4 Lymph node metastasis of early-stage ESCC is present in approximately 0% of M1 or M2 cancer, 10% of M3 or SM1 cancer, and 50% of SM2 or SM3 cancer.5 However, little is known about the mechanism of lymph node metastasis or lymphovascular invasion at the early stage of cancer progression. Before the advent of a reliable marker of lymphatic vessels, lymphatic invasion had been diagnosed using hematoxylin and eosin (H&E) staining, which reveals cancer cells floating within the endothelial-lined space. However, lymphatic invasion cannot be recognized as complete occlusion of lymphatic vessels by cancer cells because the endothelial cells are too thin to be discerned easily, and the cancer cells are recognized as cancer nests. The lymphatic endothelium is reported to show strong expression of the marker antigen D2-40,6 and many studies have reported the usefulness of immunostaining with anti-D2-40 monoclonal antibody for identification of lymphatic invasion in the cancer stroma or even cancer nests of ESCC.7–11 It has been reported that vascular endothelial growth factor C (VEGF-C) and D (VEGF-D) induce not only angiogenesis but also lymphangiogenesis.12–15 In addition, VEGF-C and VEGF-D expression is reported to be significantly correlated with lymphovascular density (LVD), lymphatic invasion, and lymph node metastasis in advanced ESCC.16–25 However, details of lymphangiogenesis at the early stage of ESCC remain unclear. In this study, we focused on lymphangiogenesis at the early stage of ESCC progression in relation to lymph node metastasis using immunostaining for D2-40, VEGF-C, and VEGF-D. MATERIALS AND METHODS Tissue samples For this study, we employed 79 samples from 59 patients who had undergone histological examinations at Saitama Medical Center, Saitama Medical University, between 2006 and 2014. The tissue samples comprised 13 specimens of normal squamous epithelium, 6 specimens of low-grade intraepithelial neoplasia (LGIN), and 60 specimens of superficial ESCC (M1: 12 lesions, M2: 12 lesions, M3: 10 lesions, SM1: 8 lesions, SM2: 4 lesions, SM3: 14 lesions). The tissue samples were obtained by esophageal biopsy (n = 3), endoscopic resection (n = 23), or esophagectomy with lymph node dissection (n = 53). The 13 samples of normal squamous epithelium were all located distantly from the cancer lesion in esophagectomy specimens. We excluded specimens from patients who had undergone radiotherapy and/or chemotherapy before lesion resection. For analysis of lymph node metastasis, we evaluated cases showing M3 or deeper cancer invasion only. We excluded five cases in which further advanced cancers coexisted and eight cases for which endoscopic resection had been performed for analysis of lymph node metastasis. Pathological diagnosis was made according to the Guidelines for Clinical and Pathologic Studies on Carcinoma of the Esophagus (10th ed.).26 Sections were cut from 3-mm-wide step-sectioned blocks of endoscopic resection specimens, or from 5-mm-wide blocks obtained from surgically resected esophagi, and stained with H&E. We divided the specimens into four different histological types, i.e. normal squamous epithelium, LGIN, M1-M2 cancer, and M3 or deeper cancer, by reference to the frequency of lymph node metastasis. The study was performed under a protocol approved by our hospital ethics committee. Immunohistochemical staining Tissue samples fixed in 10% formalin and embedded in paraffin were cut into sections 4 μm thick and mounted on slides. Immunohistochemical staining of D2-40 was performed using the streptavidin–biotin–peroxidase method. The slides were immunostained with mouse anti-D2-40 monoclonal antibody (clone D2-40 (Nichirei Bioscience Co., Tokyo, Japan); ready to use) using a Ventana Bentimark XT machine (Ventana, Tuscon, Arizona, USA). Immunostaining for VEGF-C and VEGF-D was performed with a Leica Bond-III automatic immunostainer (Leica Biosystems Newcastle Ltd., UK). The slides were immunostained with mouse anti-VEGF-C monoclonal antibody (clone F-10 (Santa Cruz Biotechnology, Texas, USA) 1:1000) and mouse anti-VEGF-D monoclonal antibody (clone #78,923 (R &D Systems, Minneapolis, USA); 1:100). Counter staining was performed using hematoxylin (Hematoxylin 3G (Sakura Finetek Japan, Tokyo, Japan)). Quantification of lymphatic vessel density On the basis of D2-40 immunostaining, LVD in the lamina propria mucosae was assessed in normal esophageal mucosa, LGIN, and M1 and M2 cancer. For M3 and submucosal cancer, we counted the lymphatic vessels at the periphery and in the center of the tumor. Sections were screened according to the method described by Weidner et al.27 Briefly, using low magnifying power, three areas of strongest D2-40 staining (hot spots) were noted. Vessel counting was performed in a ×200 field, and the mean count for three fields was determined as the LVD. Evidence of a visible lymphatic vessel lumen was not required. Quantification of VEGF-C and D Expression of VEGF-C and D in over 50% of the cells examined was considered positive. Expression of VEGF-C and D was evaluated in each of three fields in D2-40 hot spots at ×200 magnification using a light microscope. All assessments were performed by two independent investigators (YK and TT) who were blinded to the clinical data. Statistical analysis Data are expressed as median and range. Correlations between the groups were assessed by chi-squared test. Differences between groups were analyzed by Mann-Whitney U test and Kruskal-Wallis test followed by Dunn's test. Differences at P < 0.05 were considered significant. RESULTS Expression of D2-40, VEGF-C and D in esophageal lesions Immunostaining for each of D2-40, VEGF-C and D in normal squamous epithelium, M1 cancer, M2 cancer, and submucosal cancer is shown in Figure 1. D2-40 expression was detected in lymphatic endothelial cells. In the normal esophageal mucosa, lymphatic vessels were found mainly in the lamina propria mucosae. Basal layer cells in some specimens of normal epithelium showed positivity for D2-40 (Fig. 1a). In lesions of M1 and M2 cancer, hot spots of D2-40-positive lymphatic vessels were found in the lamina propria mucosae (Fig. 1b,c). In lesions of M3 or deeper cancer, hot spots were located at the periphery of the tumor, in the cancer stroma, and inside cancer nests (Fig. 1d). Fig. 1 View largeDownload slide Expression of D2-40, VEGF-C, and VEGF-D in normal squamous epithelium and esophageal cancer (counterstaining with hematoxylin). (a–d) Immunohistochemical staining for D2-40 (×100). (e–h) Immunohistochemical staining for VEGF-C (×100). i-l: Immunohistochemical staining for VEGF-D (×100). (a): Normal squamous epithelium stained for D2-40. Lymphatic vessels are visible as brown capillaries. Lymphatic vessels are frequently found in the lamina proproia mucosae rather than the submucosal layer. Basal layer cells in some cases are positive for D2-40. (b,c): M1 (b) and M2 (c) cancer lesions stained for D2-40. Lymphatic vessels are massively aggregated in the lamina propria mucosae. (d): Submucosal cancer stained for D2-40. Lymphatic invasion (arrows) can be seen in the cancer stroma. (e, i): Normal squamous epithelium stained for VEGF-C (e) and VEGF-D (i). Both VEGF-C and D are negative in normal squamous epithelium, but are weakly positive in the duct of an esophageal gland proper (arrows). (f, g): Positive immunoreactivity for VEGF-C in case of M1 (f) and M2 (g) cancer, and for VEGF-D in cases of M1 (j) and M2 (k) cancer (×100). Cancerous lesions are clearly demarcated from the normal squamous epithelium (f). (h, l): VEGF-C (h) and D (l) in submucosal cancer (×100). Homogeneously stained cancer nests can be seen. Mononuclear cells that have infiltrated the cancer stroma are also positive for VEGF-C and D. Fig. 1 View largeDownload slide Expression of D2-40, VEGF-C, and VEGF-D in normal squamous epithelium and esophageal cancer (counterstaining with hematoxylin). (a–d) Immunohistochemical staining for D2-40 (×100). (e–h) Immunohistochemical staining for VEGF-C (×100). i-l: Immunohistochemical staining for VEGF-D (×100). (a): Normal squamous epithelium stained for D2-40. Lymphatic vessels are visible as brown capillaries. Lymphatic vessels are frequently found in the lamina proproia mucosae rather than the submucosal layer. Basal layer cells in some cases are positive for D2-40. (b,c): M1 (b) and M2 (c) cancer lesions stained for D2-40. Lymphatic vessels are massively aggregated in the lamina propria mucosae. (d): Submucosal cancer stained for D2-40. Lymphatic invasion (arrows) can be seen in the cancer stroma. (e, i): Normal squamous epithelium stained for VEGF-C (e) and VEGF-D (i). Both VEGF-C and D are negative in normal squamous epithelium, but are weakly positive in the duct of an esophageal gland proper (arrows). (f, g): Positive immunoreactivity for VEGF-C in case of M1 (f) and M2 (g) cancer, and for VEGF-D in cases of M1 (j) and M2 (k) cancer (×100). Cancerous lesions are clearly demarcated from the normal squamous epithelium (f). (h, l): VEGF-C (h) and D (l) in submucosal cancer (×100). Homogeneously stained cancer nests can be seen. Mononuclear cells that have infiltrated the cancer stroma are also positive for VEGF-C and D. Normal squamous cells exhibited no or only faint staining for both VEGF-C and D. Ductal cells of esophageal glands proper in normal esophageal tissue showed positive immunoreactivity for VEGF-C and D (Fig. 1e,i). VEGF-C and D expression was evident in the cytoplasm of the cancer cells. In M1 and M2 cancer, expression of both VEGF-C and D was clearly demarcated from the adjacent normal squamous epithelium (Fig. 1f,g,j,k). In cases of M3 or deeper cancer, positivity was even, and no heterogeneity was observed in cancer nests (Fig. 1h,l). In stromal tissue, some mononuclear cells also exhibited positivity for VEGF-C and D. Correlation of histological type with lymphatic vessel density and expression of VEGF-C and D (Table 1) Table 1 Correlation between each histological type and LVD, VEGF-C and D Lymphatic vessel density† VEGF-C‡ VEGF-D‡ (Median (range)) Negative (%) Positive (%) Negative (%) Positive (%) Normal (n = 13) 7.3 (3.0–10.3) 13 (100) 0 (0) 13 (100) 0 (0) LGIN (n = 6) 11.2 (6.5–18.0) 6 (100) 0 (0) 6 (100) 0 (0) M1 and M2 (n = 24) 13.5 (5.3–34.3)§ 9 (37.5) 15 (62.5) 13 (54.2) 11 (45.8) M3 or deeper (n = 36) 14.8 (7.3–27.0)§ 11 (30.6) 25 (69.4) 20 (55.6) 16 (44.4) Lymphatic vessel density† VEGF-C‡ VEGF-D‡ (Median (range)) Negative (%) Positive (%) Negative (%) Positive (%) Normal (n = 13) 7.3 (3.0–10.3) 13 (100) 0 (0) 13 (100) 0 (0) LGIN (n = 6) 11.2 (6.5–18.0) 6 (100) 0 (0) 6 (100) 0 (0) M1 and M2 (n = 24) 13.5 (5.3–34.3)§ 9 (37.5) 15 (62.5) 13 (54.2) 11 (45.8) M3 or deeper (n = 36) 14.8 (7.3–27.0)§ 11 (30.6) 25 (69.4) 20 (55.6) 16 (44.4) †Kruskal-Wallis test P < 0.001; ‡Chi-square test P < 0.005; §Dunn test P < 0.01. LGIN, low-grade intraepithelial neoplasia; LVD, lymphatic vessel density; VEGF, vascular endothelial growth factor. View Large Table 1 Correlation between each histological type and LVD, VEGF-C and D Lymphatic vessel density† VEGF-C‡ VEGF-D‡ (Median (range)) Negative (%) Positive (%) Negative (%) Positive (%) Normal (n = 13) 7.3 (3.0–10.3) 13 (100) 0 (0) 13 (100) 0 (0) LGIN (n = 6) 11.2 (6.5–18.0) 6 (100) 0 (0) 6 (100) 0 (0) M1 and M2 (n = 24) 13.5 (5.3–34.3)§ 9 (37.5) 15 (62.5) 13 (54.2) 11 (45.8) M3 or deeper (n = 36) 14.8 (7.3–27.0)§ 11 (30.6) 25 (69.4) 20 (55.6) 16 (44.4) Lymphatic vessel density† VEGF-C‡ VEGF-D‡ (Median (range)) Negative (%) Positive (%) Negative (%) Positive (%) Normal (n = 13) 7.3 (3.0–10.3) 13 (100) 0 (0) 13 (100) 0 (0) LGIN (n = 6) 11.2 (6.5–18.0) 6 (100) 0 (0) 6 (100) 0 (0) M1 and M2 (n = 24) 13.5 (5.3–34.3)§ 9 (37.5) 15 (62.5) 13 (54.2) 11 (45.8) M3 or deeper (n = 36) 14.8 (7.3–27.0)§ 11 (30.6) 25 (69.4) 20 (55.6) 16 (44.4) †Kruskal-Wallis test P < 0.001; ‡Chi-square test P < 0.005; §Dunn test P < 0.01. LGIN, low-grade intraepithelial neoplasia; LVD, lymphatic vessel density; VEGF, vascular endothelial growth factor. View Large The median LVD (range) revealed by D2-40 staining in the normal esophageal mucosa, LGIN, M1-M2 cancer, and M3 or deeper cancer was 7.3 (3.0–10.3), 11.2 (6.5–18.0), 13.5 (5.3–34.3), and 14.8 (7.3–27.0), respectively. The median LVD based on D2-40 immunostaining was lowest in the normal esophageal mucosa, followed in ascending order by LGIN, M1-M2 cancer, and M3 or deeper cancer, and the difference was significant by Kruskal-Wallis test (P < 0.001). Post hoc Dunn's test revealed that the LVD for M1-M2 cancer and M3 or deeper cancer was significantly higher than that for normal squamous epithelium (P < 0.001). Expression of both VEGF-C and D was not considered positive in normal squamous epithelium and LGIN. In LGIN, weak expression of VEGF-C and D was found in 4/6 and 2/6 lesions, respectively, but less than 50% of cells were positive. High expression of VEGF-C and D was observed in M1 and M2 cancer (62.5% and 45.8%, respectively) and in M3 or deeper cancer (69.4% and 44.4%, respectively). Chi-squared test showed that the expression of VEGF-C and D was significantly correlated between the groups (P < 0.005). Correlation between LVD and expression of VEGF-C or D in ESCC Figure 2 shows the correlation between LVD and the expression of VEGF-C or D in ESCC. The median (range) LVD was 15.2 (5.3–34.3) in VEGF-C-positive cases and 11.3 (6.3–23.0) in VEGF-C-negative cases, the difference being significant (P < 0.001). Similarly, the median (range) LVD was 17.0 (9.0–34.3) in VEGF-D-positive cases and 13.0 (5.3–23.0) in VEGF-D-negative cases, the difference also being significant (P < 0.001). Fig. 2 View largeDownload slide Lymphatic vessel density in relation to positivity or negativity for VEGF-C and D. (a) VEGF-C (median (range)). Negative: 11.3 (6.3–23.0), Positive: 15.2 (5.3–34.3), Mann-Whitney U test P < 0.005. (b) VEGF-D (median (range)). Negative: 13.0 (5.3–23.0), Positive: 17.0 (9.0–34.3), Mann-Whitney U test P < 0.005. Fig. 2 View largeDownload slide Lymphatic vessel density in relation to positivity or negativity for VEGF-C and D. (a) VEGF-C (median (range)). Negative: 11.3 (6.3–23.0), Positive: 15.2 (5.3–34.3), Mann-Whitney U test P < 0.005. (b) VEGF-D (median (range)). Negative: 13.0 (5.3–23.0), Positive: 17.0 (9.0–34.3), Mann-Whitney U test P < 0.005. Correlation between expression of VEGF-C and D, LVD, and lymphatic invasion or lymph node metastasis (Table 2) Table 2 Correlation between lymphatic invasion or lymph node metastasis and VEGF-C, D, and LVD VEGF-C VEGF-D Lymphatic vessel density Negative Positive Negative Positive (median (range)) Lymphatic invasion  Negative 9 14 15 8 13.7 (7.3–27.0)  Positive 2 11 5 8 18.7 (9.0–25.7)† P = 0.14 P = 0.12 Lymph node metastasis  Negative 3 8 5 6 13.0 (7. 7–25.3)  Positive 3 9 4 8 18.2 (13.0–27.0)† P = 0.90 P = 0.55 VEGF-C VEGF-D Lymphatic vessel density Negative Positive Negative Positive (median (range)) Lymphatic invasion  Negative 9 14 15 8 13.7 (7.3–27.0)  Positive 2 11 5 8 18.7 (9.0–25.7)† P = 0.14 P = 0.12 Lymph node metastasis  Negative 3 8 5 6 13.0 (7. 7–25.3)  Positive 3 9 4 8 18.2 (13.0–27.0)† P = 0.90 P = 0.55 †Mann-Whitney U test P < 0.05. View Large Table 2 Correlation between lymphatic invasion or lymph node metastasis and VEGF-C, D, and LVD VEGF-C VEGF-D Lymphatic vessel density Negative Positive Negative Positive (median (range)) Lymphatic invasion  Negative 9 14 15 8 13.7 (7.3–27.0)  Positive 2 11 5 8 18.7 (9.0–25.7)† P = 0.14 P = 0.12 Lymph node metastasis  Negative 3 8 5 6 13.0 (7. 7–25.3)  Positive 3 9 4 8 18.2 (13.0–27.0)† P = 0.90 P = 0.55 VEGF-C VEGF-D Lymphatic vessel density Negative Positive Negative Positive (median (range)) Lymphatic invasion  Negative 9 14 15 8 13.7 (7.3–27.0)  Positive 2 11 5 8 18.7 (9.0–25.7)† P = 0.14 P = 0.12 Lymph node metastasis  Negative 3 8 5 6 13.0 (7. 7–25.3)  Positive 3 9 4 8 18.2 (13.0–27.0)† P = 0.90 P = 0.55 †Mann-Whitney U test P < 0.05. View Large Lymphatic invasion was found in 13 of 36 M3 or deeper lesions (36.1%; M3: 0/10, SM1:3/8, SM2:1/4, SM3:9/14), whereas no lymphatic invasion was found in M1 and M2 lesions. Analysis of the M3 or deeper group revealed no correlation between the expression of VEGF-C or D and lymphatic invasion (P = 0.14 and 0.12, respectively). However, the median (range) LVD in cases positive for lymphatic invasion (18.7 (9.0–25.7)) was significantly higher than in cases that were negative (13.7 (7.3–27.0)) (P = 0.02). Lymph node metastasis was found in 12 of 23 cases of surgically resected M3 or deeper cancer (52.2%; M3: 1/3, SM1:0/4, SM2:1/3, SM3:10/13). We also found no correlation between VEGF-C or D expression and lymph node metastasis (P = 0.90 and 0.55, respectively). The median (range) LVD in cases that were positive for lymph node metastasis (18.2 (13.0–27.0)) was significantly higher than in cases that were negative (13.0 (9.0–25.3)) (P = 0.03). DISCUSSION Understanding the changes that occur in lymphatic vessels during cancer progression would undoubtedly help to clarify the mechanisms of lymph node metastasis and carcinogenesis. Accurate knowledge of the dynamic changes at the early stage of cancer progression might provide clues to chemoprevention, molecular targeting therapy, or early detection of cancer. Lymphatic invasion is well known to be a useful marker for prediction of lymph node metastasis.7,9–11 D2-40 has been shown to be a selective marker of the lymphatic endothelium and facilitates the objective identification of lymphatic invasion in comparison with conventional HE staining.10 In addition, D2-40 immunostaining makes it possible to assess the degree of lymphangiogenesis based on the LVD count. It has been reported that LVD is associated with lymph node metastasis and prognosis of gastric cancer, SCC of the uterine cervix, and head and neck SCC.28–30 The use of D2-40 immunostaining has shown that 0–5% of cases of M1 and M2 ESCC are positive for lymphatic invasion, whereas 35–60% of cases of M3 or deeper cancer are positive.5,10 These results are in agreement with our present findings. Similarly, some previous studies found no lymph node metastasis in cases of M1 and M2 ESCC, whereas 25–40% of M3 or deeper cases were positive.5,9,10 Our finding that 52.2% of cases of M3 or deeper ESCC were positive for lymph node metastasis suggested a higher incidence than that reported previously.5,9,10 One of the reasons for this high incidence of lymph node metastasis may have been that we excluded eight cases of M3 or deeper cancer treated using endoscopic mucosal resection. None of these cases treated by endoscopic resection showed clinical evidence of lymph node metastasis or distant metastasis at least two years after treatment. If these cases had been counted as lymph node metastasis negative, the incidence of lymph node metastasis would have been 38.7%. VEGF, especially VEGF-A, is known as to be a major inducer of angiogenesis and vessel permeability.31 Five additional members of the VEGF family—VEGF-B, VEGF-C, VEGF-D, VEGF-E, and placenta growth factor (PIGF)—have been characterized.32 Among them, VEGF-C and D are known to induce both angiogenesis and lymphangiogenesis.13,15 These cytokines act as ligands for vascular endothelial growth factor receptor (VEGFR)-2, which is expressed on vascular endothelial cells and is essential for formation of blood vessels.33 In addition, both cytokines can bind to VEGFR-3, which is expressed predominantly in the endothelium of lymphatic vessels.34 Thus, VEGF-C and D are reported to be the main inducers of lymphangiogenesis in various malignant tumors, including ESCC.14,16–25 The immunohistochemical positivity rate for VEGF-C is reported to be 40–100% in ESCC.14–24 With regard to VEGF-D in ESCC, two articles have reported positivity rates of 73.1% and 65.9%.24,25 Only one article has reported the positivity rate for both VEGF-C and D in ESCC. In that report, the VEGF-C positivity rate in cases of esophageal neoplasia (including esophageal dysplasia) was higher than the positivity rate for VEGF-D.24 This finding is similar to that in our present report. However, these previous studies14–25 included only a limited number of superficial ESCC cases, and thus the VEGF-C and D status at the early stage of ESCC progression was unclear. In this study, we examined 60 lesions of superficial ESCC and 6 lesions of LGIN. We divided the cancer lesions into two groups (i.e. M1 and M2 cases vs. M3 or deeper cases) in accordance with the reported rate of lymph node metastasis. The positivity rates for VEGF-C and D in M1 and M2 cases were 62.5% and 45.8%, respectively, being similar to the rate in M3 or deeper cases. On the other hand, all of the specimens of normal squamous epithelium and LGIN were judged as negative for both VEGF-C and D. In addition, LVD in M1 and M2 cases was double that for normal squamous epithelium, and the difference between the two groups was significant. LVD in M3 or deeper cases was slightly higher than that in M1 and M2 cases, and the difference was not significant. In addition, cases of ESCC positive for both VEGF-C and D showed a significantly higher LVD (P < 0.001) than cases that were negative, suggesting that both VEGF-C and D may play important roles in the recruitment of lymphatic ducts, and that lymphangiogenesis is activated at the very early stage of carcinogenesis. VEGFR-3, as well as VEGF-C, reveals significant correlation with LVD in ESCC.16 Overexpression of the VEGF-C may induce tumor lymphangiogenesis by stimulating VEGFR-3 expression on lymphatic endothelial cells. These findings will be a hint of further study. It has been reported that expression of VEGF-C and D is significantly correlated with lymphatic invasion, lymph node metastasis, and prognosis in various types of cancer including ESCC.16–25 Omoto et al.16 investigated lymphangiogenesis of ESCC (both superficial and advanced) in detail using immunohistochemistry for VEGF-C, VEGFR-3, and D2-40, and found that VEGF-C positivity was significantly correlated with lymphatic invasion, lymph node metastasis, and LVD. On the other hand, Noguchi et al. reported that VEGF-C expression showed no correlation with lymphatic invasion, lymph node metastasis, and prognosis. This study revealed that LVD was significantly higher in M3 or deeper cancer lesions with lymphatic invasion and lymph node metastasis than in those negative for lymphatic invasion and lymph node metastasis. However, no significant correlation was found between the expression of VEGF-C or D and lymphatic invasion or lymph node metastasis. In addition, despite the high rate of VEGF-C and D positivity and high level of LVD in M1-M2 cancers (similar to M3 or deeper cancers), none of the cases showed lymphatic invasion or lymph node metastasis. Therefore, other factors associated with induction of lymphatic invasion and subsequent lymph node metastasis may exist. Overall, the present findings suggest that cancer cells produce VEGF-C and D from the very early stage of ESCC progression. VEGF-C and D activate lymphangiogenic switching, but may not induce lymphatic invasion and lymph node metastasis directly. These data suggest that the increase in the number of lymphatic vessels leads to lymphatic invasion and subsequent lymph node metastasis at the early stage of ESCC progression. Acknowledgment This study was supported by MEXT KAKENHI Grant number 26461047. We are grateful to Tomoyuki Kawada for his kind advice and confirmation of statistical analysis. Human rights statement and informed consent All procedures followed were in accordance with the ethical standards of the committees responsible for human experimentation (institutional and national) and with the Helsinki Declaration of 1964 and later versions. Informed consent for inclusion of tissue samples in this study was obtained from all patients or their representatives. Notes All of the authors who are listed in the title page contributed significantly to this paper, and match the authorship that is stated in the “Guidelines of the international committee of Medical Journal Editors.” Conflict of interest disclosure: Youichi Kumagai received a research grant from MEXT KAKENHI. References 1 The Japan Esophageal Society . Japanese classification of esophageal cancer, tenth edition: part I . Esophagus 2009 ; 6 : 1 – 25 . CrossRef Search ADS 2 Enzinger P C , Mayer R J . Esophageal cancer . N Engl J Med 2003 ; 349 : 2241 – 52 . Review . 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Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during development . Proc Natl Acad Sci 1995 ; 92 : 3566 – 70 . Google Scholar CrossRef Search ADS PubMed © The Authors 2018. Published by Oxford University Press on behalf of International Society for Diseases of the Esophagus. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Diseases of the EsophagusOxford University Press

Published: Aug 1, 2018

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