Angiogenesis and lymphangiogenesis in stage 1 germ cell tumours of the testis.

OBJECTIVE: To determine whether angiogenesis can be used as an additional prognostic indicator in patients with stage 1 germ cell tumours of the testis. PATIENTS AND METHODS: Paraffin sections were assessed immunohistochemically from 51 patients with clinical stage 1 germ cell tumours of the testis (28 seminoma, 23 teratoma) treated by orchidectomy and surveillance only. Sections were analysed for microvascular density (MVD), and expression of the angiogenic factors vascular endothelial growth factor (VEGF) and thymidine phosphorylase (TP). In addition, in the seminoma cases the presence of mRNA for the lymphangiogenic factor VEGF-C was examined by in situ hybridization, and its corresponding receptor VEGFR-3 by immunohistochemistry. RESULTS: Teratoma specimens had a significantly higher mean (range) MVD (85, 26-163; P < 0.01) than both seminoma (37, 16-91) and four normal specimens (26, 18-30). Teratoma specimens also had significantly higher VEGF expression than both seminoma and normal specimens (P < 0.01). Despite these differences between groups, and indeed individual tumours, there was no significant correlation between MVD and VEGF, or between either MVD or VEGF and relapse-free survival. TP expression was significantly greater in tumours than in normal specimens (P < 0.02) but with very little inter-tumour variation. VEGF-C mRNA and VEGFR-3 protein were detected in a third to a half of cases, with expression mostly around endothelial vessels. CONCLUSIONS: The marked differences between normal testis and tumours implicate angiogenesis in the biology of germ cell tumours of the testis. In addition, the detection of factors involved in lymphangiogenesis in some seminomas, tumours which initially metastasize primarily to lymph nodes, indicate that although not prognostic in this study, further studies are warranted in both these areas in the search for further prognostic indicators and therapeutic targets.

Expression of vascular endothelial growth factor in patients with testicular germ cell tumors as an indicator of metastatic disease.

BACKGROUND: Angiogenesis is essential for tumor growth and metastasis. Vascular endothelial growth factor (VEGF) and thymidine phosphorylase (TP)/platelet-derived endothelial cell growth factor (PD-ECGF) are involved in increased angiogenic activity and disease progression in solid tumors. However, there is no information regarding the association of these angiogenic factors with clinicopathologic findings in testicular germ cell tumors (GCTs). METHODS: The authors examined the expression of VEGF and TP as well as microvessel density in GCTs and their association with clinicopathologic findings. Expression of VEGF and TP and microvessel density were examined immunohistochemically in 80 GCTs, including 33 seminomas (25 tumors with organ-confined disease and 8 with metastasis) and 47 nonseminomatous testicular GCTs (NSGCTs) (20 tumors with organ-confined disease and 27 with metastasis). Expression of VEGF also was examined in four GCTs and one nonneoplastic testis by immunoblotting. RESULTS: VEGF protein was expressed more highly in GCTs compared with nonneoplastic testes. VEGF expression in GCTs was correlated significantly with microvessel count (P < 0.001). Both VEGF expression and microvessel count were correlated with metastasis in seminoma (P = 0.008 and P < 0.001, respectively), but only VEGF expression was identified as statistically significant by multiple regression analysis (P = 0.006). Conversely, four variables (VEGF expression, microvessel count, the presence of venous invasion, and the presence of embryonal carcinoma elements in the primary tumor) were correlated with metastasis in NSGCT (P < 0.001, P < 0.001, P = 0.004, and P = 0.029, respectively). However, multiple regression analysis revealed that only VEGF expression and microvessel count were significant factors for metastasis (P < 0.007 and P < 0.001, respectively). In contrast, high levels of TP were observed in infiltrating cells, but not in the majority of cancer cells. CONCLUSIONS: The findings of the current study suggest that VEGF expression is involved in tumor development, angiogenesis, and metastasis in GCT.

Vascular density does not predict future metastatic disease in clinical stage 1 non-seminomatous germ cell tumours of the testis.

AIM: This study aimed to determine whether patients with stage 1 testicular non seminomatous germ cell tumours (NSGCT) with high vascular density have a greater risk of disease recurrence than those with a low vascular density. METHODS AND RESULTS: Orchidectomy specimens from 42 patients with stage 1 NSGCT, treated by orchidectomy and surveillance alone, were studied. Vessel density was counted in tumour sections immunohistochemically stained for CD34. The mean of the three highest counts (x250, field size 0.67 mm2) for each tumour was used. Tumour vessel density was very similar for relapsing and non relapsing patients. Vascular invasion was the only variable significantly predictive of disease recurrence at 2 years post-orchidectomy (P = 0.025). There was wide variation of vessel counts between different blocks of a tumour, compared with interobserver variation. The tumour tissue type in the area of highest vessel density was embryonal carcinoma in 50% and teratoma (mature or immature) in 38%. CONCLUSIONS: We confirmed the value of vascular invasion as a prognostic marker in stage 1 NSGCT. Tumour vessel density was of no prognostic value. Two factors may contribute to this. First, there was wide variation of vessel density between different blocks of a tumour. Second, the most vascular area in a tumour was frequently in low-grade tumour.

Neovascularization in human germ cell tumors correlates with a marked increase in the expression of the vascular endothelial growth factor but not the placenta-derived growth factor.

Neoangiogenesis is a prerequisite for tumor growth and metastasis. In germ cell cancer patients with the disease limited to the testicle (stage A), tumor-associated neovascularization is predictive of metastatic disease (stage B). To investigate the molecular mechanisms underlying neovascularization in human germ cell tumors (GCTs), we analysed the expression of two angiogenic growth factors, vascular endothelial growth factor (VEGF) and placenta growth factor (P1GF), and of their receptors (FLT-1) and Flk-1/KDR) in a panel of testicular tumors. In this study we show a marked increase in VEGF expression in 36/44 (81.8%) primary testicular-derived GCTs, as compared to normal testis, that significantly correlates with a high density of intratumor microvessels (r = 0.72461, P < 0.001; n = 24). As determined by RT - PCR and/or Western blot, the predominant VEGF isoforms expressed in GCTs are the VEGF121 and VEGF165, which are more efficiently secreted by the cells, and thus more active in eliciting angiogenesis. Conversely, in the case of PIGF, only a weak correlation with the vascular density of tumors is observed (r = 0.26599, P < 0.05; n = 24). Northern blot analysis also revealed significant up-regulation of VEGF/ PIGF receptors in highly vascularized germ cell tumors, compared to normal testes. These findings suggest that VEGF may act in a paracrine manner to induce neovascularization, oedema extravasation and cyst formation in human germ cell tumors. The correlation between VEGF expression and the vascular density of tumors, suggest that the evaluation of VEGF expression may be of help in predicting patients at risk for metastatic diseases. Finally, we demonstrate that VEGF up-regulation may occur at the RNA level since no gene amplification is observed; conversely, in in vitro models such as the embryonal stem cell line NTERA-2 and the choricarcinoma JEG-3 cell line, VEGF (but not PIGF) mRNA expression is regulated by hypoxic stress.

Prognostic significance of immunohistochemical proliferation markers (Ki-67/MIB-1 and proliferation-associated nuclear antigen), p53 protein accumulation, and neovascularization in clinical stage A nonseminomatous testicular germ cell tumors.

Histopathologic features alone fail to reliably stratify patients with clinical Stage A nonseminomatous germ cell tumors of the testis into groups with high and low risk for occult metastatic disease. Previous flow cytometric studies at Indiana University demonstrated a significant correlation between high proliferative activity and metastatic disease. The current study evaluated the prognostic significance of immunohistochemical markers related to tumor proliferation and aggressiveness in a consecutive series of clinical Stage A nonseminomatous germ cell tumors patients who underwent retroperitoneal lymph node dissection. Archival material of the orchiectomy specimens of 62 patients (45 pathologic Stage A, 17 with metastatic disease) was reviewed and immunohistochemically stained for Ki-67 antigen (MIB-1), proliferation-associated nuclear antigen (PC10), p53 protein (Pab1801), and Factor-VIII-related antigen (neovascularization). Staining with MIB-1 was significantly higher in the metastatic group (mean 80.2%, standard deviation [SD] 15.5) than in pathologic Stage A cases (66.3%, SD 27.9; P = 0.0032) and was predictive of metastatic status with a sensitivity of 82% and specificity of 69%. In this study, no patient with a MIB-1 value less than 52% had metastases. Proliferation-associated nuclear antigen and p53 staining correlated with MIB-1 values (R = 0.63 and 0.55, respectively) but did not correlate with metastatic status. Tumor angiogenesis was also not predictive of metastatic status. Assessment of proliferation rates using MIB-1 antibody in clinical Stage A nonseminomatous germ-cell-tumor patients may prove helpful in predicting metastatic status.(ABSTRACT TRUNCATED AT 250 WORDS)

Neovascularization in clinical stage A testicular germ cell tumor: prediction of metastatic disease.

Increased numbers of blood vessels (angiogenesis or neovascularization) in certain primary tumors correlates with an increased risk for metastatic disease. We therefore conducted a blinded review of the resected testicular germ cell tumors of 65 clinical stage A patients to evaluate the usefulness of angiogenesis in identifying those patients with clinically occult nodal metastases (pathological stage B). Angiogenesis was assessed in the primary tumors using an immunohistochemical stain for factor VIII-related antigen assay for quantitation of microvessel counts. Of 65 clinical stage A patients, 43 had pathological stage B disease at retroperitoneal lymph node dissection. Eleven patients had microvessel counts > 30 microvessels/x 400 high powered field, and all of these patients had pathological stage B disease (P = 0.02 in univariate analysis). Multiple regression analysis using microvessel count and other histological findings found to be prognostic (venous invasion, lymphatic invasion, presence of embryonal carcinoma, and absence of yolk sac tumor) showed that only the absence of a yolk sac tumor component was significantly predictive of occult metastases. This study shows that angiogenesis, as measured by quantitation of microvessel counts in the primary tumor of germ cell neoplasms, is significantly predictive of occult nodal metastatic disease by univariate analysis in clinical stage A patients. The prospective use of angiogenesis quantitation needs to be defined.

Vascular considerations in postchemotherapy. Retroperitoneal lymph-node dissection: Part II.

A total of 710 patients underwent postchemotherapy retroperitoneal lymph-node dissection (RPLND) from 1965 to 1992. Ten patients, all with bulky disease, required aortic replacement either postoperatively (n = 3) or during RPLND (n = 7). The principal risk factor for aortic rupture after RPLND was an extended subadventitial aortic dissection made necessary by tumor fixation. Also, duodenal enterotomy or extensive violation of the bowel serosa was a further risk for aortoenteric fistula. Prospective aortic grafting may be indicated in the presence of these risk factors. In our four elective cases, there was no further vascular or bowel complication. Omental interposition further protects against fistula formation. Although rarely indicated except under the most extenuating circumstances, the exposure requirements of RPLND permit aortic grafting as a relatively straightforward procedure that is feasible and well tolerated in this small subset of patients.

Vascular considerations in postchemotherapy. Retroperitoneal lymph-node dissection: Part I--Vena cava.

A total of 42 patients underwent inferior vena cava resection (n = 40) or intraluminal tumor thrombectomy (n = 2) during retroperitoneal lymph-node dissection (RPLND) for bulky abdominal metastatic nonseminomatous germ-cell cancer (7% of all postchemotherapy RPLND cases). The three indications for caval resection included tumor clearance (38%), caval scar occlusion (14%), and caval tumor thrombus (48%). En bloc caval resection to achieve tumor clearance was justified by subsequent nodal pathology (cancer in 63% of specimens, teratoma in 31% specimens). Caval resection in the presence of scar occlusion was required de facto by virtue of its incorporation in the specimen. Caval resection or thrombectomy is indicated for intraluminal tumor thrombus because thrombus pathology (cancer, 35%; teratoma, 45%; fibrosis, 20%) reflected nodal pathology in 71% of cancer cases, 78% of teratoma cases, and 100% of fibrosis cases. The complications of caval resection were generally transitory. The 71% survival rate justifies this intensive surgical approach because these patients had exhausted all chemotherapeutic options.