1. Introduction
Cervical neoplasia remains one of the most controversial issues for clinicians, pathologists and researchers. Screening programs have reduced the incidence of invasive neoplastic lesions but have not changed the rate of precursor lesions. Human papilloma virus (HPV) is confirmed to be involved in the etiology of uterine cervix lesions. Recently, Coutlée
Uterine cervical lesions and the involvement of LV in the tumor development, progression and metastasis still remains a field of debate. Few articles describe lymphatic vessel density and the potential role of lymphangiogenesis in the progression of cervical neoplasia[7-8]. The relationship between lymphangiogenesis and the invasion of cervical cancer was suggested by Hashimoto
Based on the controversial data concerning expression of CD105 and Ki-67 in endothelial cells from uterine cervix lesions, the aim of the present study was to investigate the distribution and co-localization of mentioned markers in benign and malignant conditions of the uterine cervix. Concerning early steps of lymphangiogenesis, the purpose was to evaluate morphology, distribution, lymphatic microvessel density and lymphatic proliferation in different stages of cervical lesions. Comparison of type-specific features of lymphangiogenesis in premalignant and malignant lesions, correlated with proliferative status of lymphatic endothelial cells, could help detect the early lymphangiogenesis in pathological conditions of the uterine cervix. A complete characterization of the morphological and immunohistochemical data of LV could have a prognostic and therapeutic impact and confirm diagnosis in cervical lesions.
2. Morphologic and immunohistochemical methods for blood and lymphatic vessels assessement in cervical neoplasia
One of our research included 128 retrospective targeted biopsies of the uterine cervix and specimens taken from conization in patients with macroscopically detectable lesions. Specimens were fixed in buffer formalin and embedded in paraffin, based on the conventional histological technique. Step sections, 5 mm thick, were prepared for each case. Initial sections were stained with HE, for the pathological diagnosis and grade of the tumor. Lesions were stratified as follows: squamous cell metaplasia (
3. Tumor blood vessels activation and proliferation is different for each histopathologic type of cervical neoplasia
CD105 expression was absent in all cases of normal cervix. CD105 imunoreaction was restricted to the vascular activated endothelium from dysplasia, carcinoma
4. Lymphangiogenesis and lympahtic vessels in cervical neoplasia- early development with a potential prognostic impact
Basal cells of the epithelium from normal exocervix and cervical squamous metaplasia were positive for D2-40 as a continuous layer over the lamina propria of the cervix. This distribution was also observed in LSIL. A lower intensity of immunostaining was detected for basal cells of HSIL. A discontinuous pattern of the basal layer or clusters of positive cells also characterized HSIL. There were differences concerning the distribution and density of LV between the histopathological types of cervical lesions included in our study. In the normal cervix we found large LV, round or oval in shape without branches, found deep in the lamina propria, far from the stratified squamous epithelium. In the normal cervix, D2-40-positive lymphatic vessels had no positive reaction for Ki67 in endothelial cells. The same pattern was found in squamous metaplasia of the uterine cervix. Lymphatic microvascular density (LMVD) ranged between 4.8 and 6.6 vessels/200 magnification, with an average of 5.8 in the normal cervix. In squamous cell metaplasia the number of LV was not significantly different from results found in the normal cervix. A significant increase in the number of LV was found in cases with HSIL and
Expression of Prox1, VEGF-C and VEGFR3 was different concerning the histopathology of cervical lesions. Prox1 was restricted to the lymphatic and venous endothelial cell nuclei, whereas VEGF-C had a wide expression in the tumor, lymphatic endothelial and scaterred stromal cells. VEGFR3 had a strong expression in lymphatic endothelial cells from peritumoral or intratumoral lymphatic vessels, and also in the intravascular tumor emboli from invasive carcinoma cases.
Prox1 analysis showed the presence of positive reaction from CIN2 lesions (33,3% from total cases) to CIN3- 91,6% and microinvasive carcinoma – 14 cases (87,5%). Prox1 positive endothelial cells circumscribed the lumen of the lymphatic vessels in close vicinity with the epithelial proliferation of uterine cervix “
VEGF-C expression was found to be positive in all types of cervical lesions. Intensity of VEGF-C expression and number of positive cases increased from squamous metaplasia to invasive carcinoma. VEGF-C was highly expressed in tumor cells, less and inconstant in stromal cells and lymphatic vessel endothelial cells., We obtained a significant correlation between intermediate grade of VEGF-C expression and Prox1 nuclear density (p=0.044). Tumor cells were negative for Prox-1 in 100% of cases. No positive reaction was found in normal specimens, squamous metaplasia, low grade intraepithelial neoplasia or invasive carcinoma.
5. Angiogenesis and lymphangiogenesis controversies in cervical neoplasia-review of the literature
Benign lesions has not been studied as much with regard to CD105 expression. We observed a high CD105 expression in vessels from cervical metaplasia and dysplasia, and absence of endothelial proliferation was quantified using Ki-67 marker. During the early stages of tumor development TGF-b acts as an inhibitor of neoplastic proliferation. When tumor cells escape from the anti-mitotic signal exerted by TGF-b, they secrete large amount of TGF-b, promote cell invasion and metastasis and create an optimal microenvironment for promoting neo-angiogenesis[2,13]. Also, it is known that as cells progress toward fully malignant tumor cells, they undergo changes that result in reduced expression of TGF-b receptors, increased expression of TGF-b ligands, and resistance to inhibition of growth by TGF-b. The present findings suggest the same changes in the endothelial cells of activated vessels from premalignant lesions and invasive carcinoma of the uterine cervix on expression of CD105 (co-receptor for TGF-receptors I and III) without Ki-67—in cervical metaplasia and dysplasia activated vessels— and lack of CD105 staining in Ki-67-positive endothelial cells from tumor vessels of cervical invasive carcinoma. This is sustained by significant correlation found by our team between low tumor cell proliferative index (<5%) and absence of CD105 expression in the vascular bed from cervical invasive carcinoma. Many studies associated CD105 immuno-expression with proliferating endothelial cells[14-16]. The present data showed that in CD105-positive endothelial cells from premalignant and malignant lesions of the uterine cervix, activation and proliferation are two distinct processes in most of the cases. In conclusion, activation of endothelial cells is an early event that predominates in benign and premalignant conditions of the uterine cervix, while endothelial cell proliferation is observed in tumor vessel endothelial cells from invasive carcinoma of the uterine cervix.
Using the method of step sections, Roche and Norris[17] and later Leman
Prox1 has been extensively studied, mainly in the context of embryonic development of lymphatic vasculature, liver, pancreas, lens fibers cells and progenitor cells of retinal photoreceptor neurons. ). In our study, no Prox1 positive immunostaining was found for normal cervix epithelium or dysplastic one. Up to now, there are no data about Prox1 expression in tumors derived from stratified squamous epithelia. Based on our results, we assume that Prox-1 is not involved in the uterine cervix epithelial carcinogenesis as previously described for other types of neoplasia. Committment of endothelial cells through a lymphatic phenotype by its Prox1 expression appear early in the development of cervical cancer. A high number of Prox1 positive cells was observed starting from the intraepithelial neoplastic stages (CIN2 and CIN3) to microinvasive carcinoma. These findings can be partially explained by previously published data concerning lymphangiogenesis. all Prox-1 positive lymphatic vessels were positive for VEGFR-3. Similar results were previously reported in lymphangiomas by Wilting et al. Furthermore, similar Prox1 and VEGFR-3 positivity of venous endothelial cells might be evidence for the origin of lymphatic endothelial cells from venous endothelium as a main lymphangiogenic mechanism during cervical neoplasia progression. No similar data has been previously reported in the uterine cervix cancer. Thus, we consider that in cervical neoplasia, venous endothelial cells could specify through a lymphatic phenotype, by activation of Prox1 expression during tumor progression, being able to give rise to lymphatic endothelial cells. The presence of stromal Prox1 positive cells found in the present study suggests the existence of a stromal pool of cells capable of acquiring the lymphatic phenotype.
In summary, the present findings suggests by microscopy, immunohistochemistry and statistical analysis that angiogenesis and lymphangiogenesis are early events in the lesions of the uterine cervix before an overt invasion certified by morphological and immunohistochemical tests. This explains the early development of metastasis from microinvasive carcinoma stage, aggressiveness and poor response to the therapy of cervical invasive carcinoma. Further studies are necessary to evaluate angiogenesis and lymphangiogenesis in preneoplastic lesions of the uterine cervix to show their value for prognosis and therapeutic management.
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