Prognostic significance of peritumoral lymphatic vessel density and vascular endothelial growth factor receptor 3 in invasive squamous cell cervical cancer

Abstract

Cervical cancer is known to metastasize primarily by the lymphatic system. Dissemination through lymphatic vessels represents an early step in regional tumor progression, and the presence of lymphatic metastasis is associated with a poor prognosis. In patients who have undergone a radical hysterectomy, lymphovascular space invasion (LVSI), assessed on hematoxylin and eosin-stained slides, is a major factor for adjuvant therapy in patients with cervical cancer. With the advent of a lymphatic endothelial cell-specific marker, such as D2-40, it is now possible to distinguish between blood and lymphatic space invasion (LSI). In this study, the utility of D2-40 was assessed for the detection of lymphatic vessel density (LVD) and identification of LSI. The expressions of vascular endothelial growth factor receptor-3 (VEGFR-3), VEGF-C, tyrosine receptor kinase-2, and angiopoietin-1 were assessed by immunohistochemical methods on 50 patients with squamous cell carcinoma of the cervix. Clinicopathologic characteristics, including pelvic lymph node metastasis, were correlated with the above histochemical findings. We found that lymphangiogenesis, measured by an increase in peritumoral LVD, was significantly associated with positive lymph node status (P < .005). VEGFR-3 expression was significantly associated with LVD (P < .05). D2-40 staining verified LSI (P = .03) and surpassed that of hematoxylin and eosin-identified LVSI (P = .54). In conclusion, lymphangiogenic markers, specifically LVD quantified by D2-40 and VEGFR-3, are independently associated with LSI and lymph node metastasis in patients with early squamous cell carcinoma of the cervix treated with radical hysterectomy and pelvic lymphadenectomy.

Figure 1

Designation of peritumoral regions. Aperio Scan Scope (Aperio Technologies) image of D2-40-stained formalin-fixed, paraffin-embedded squamous cell carcinoma of the cervix. Green line directly adjacent to tumor mass designates tumor boundary. Peritumoral region 1 was set as the region spanning 1 mm from the tumoral border, designated by the yellow line. Peritumoral region 2 spanned 1 mm from the boarder of region 1, designated by the orange line. s indicates stroma; t, tumor. Original magnification, x2; scale bar, 1 mm.

Figure 2

Utility of D2-40 immunohistochemical staining. (A) Typical results of immunostaining with the D2-40 antibody, brown staining selectively present in lymphatic endothelium. (B) Commonly seen retraction artifact mimicking LVSI. (C) D2-40-stained endothelial cells, stained brown, verify the presence of true LSI of the cancer cells. Without the use of the lymph epithelial-specific immunohistochemistry staining, differentiation between panels A and B would prove difficult and subjective. Original magnification, x20.

Figure 3

Peritumoral LVD in cervical squamous cell carcinoma. (A) LVD in peritumoral region 1, when compared with region 2, was found to be 1.25-fold higher. (B and C) Comparison of peritumoral LVD in region 2 (B) and region 1 (C) with the absence and presence of lymph node metastasis. Lines indicate median VEGFR-3 immunostaining score values. *P < .05; ***P = .0005, ANOVA.

Figure 4

Immunohistochemical expression of VEGFR-3 in invasive cervical squamous cell carcinoma. VEGFR-3 immunopositivity was found in the cytoplasm of the tumor cells. Expression is heterogeneous within the tumor yet variable between patients. VEGFR-3 expression was graded from 1+ (faint staining; A) to 4+ (intense staining; B). Intensity of staining was found to have a statistically significant positive correlation with lymph node metastasis (P < .05, ANOVA). s indicates stroma; t, tumor. Original magnification, x40.