Vascular endothelial growth factor (VEGF)-A inhibitors exhibit unseen high responses and toxicity in recurrent epithelial ovarian cancer suggesting an important role for the VEGF/VEGFR pathway. We studied the correlation of VEGF signalling and AKT/mTOR signalling. Using a tissue microarray of clinical samples ( N =86), tumour cell immunohistochemical staining of AKT/mTOR downstream targets, pS6 and p4E-BP1, together with tumour cell staining of VEGF-A and pVEGFR2 were semi-quantified. A correlation was found between the marker for VEGFR2 activation (pVEGFR2) and a downstream target of AKT/mTOR signalling (pS6) ( R =0.29; P =0.002). Additional gene expression analysis in an independent cDNA microarray dataset ( N =24) showed a negative correlation ( R =−0.73, P <0.0001) between the RPS6 and the VEGFR2 gene, which is consistent as the gene expression and phosphorylation of S6 is inversely regulated. An activated tumour cell VEGFR2/AKT/mTOR pathway was associated with increased incidence of ascites ( χ 2 , P =0.002) and reduced overall survival of cisplatin–taxane-based patients with serous histology ( N =32, log-rank test, P =0.04). These data propose that VEGF-A signalling acts on tumour cells as a stimulator of the AKT/mTOR pathway. Although VEGF-A inhibitors are classified as anti-angiogenic drugs, these data suggest that the working mechanism has an important additional modality of targeting the tumour cells directly.

In malignant melanoma (MM) there is an urgent need to identify new markers with predictive value superior to the traditional clinical and histological parameters. Angiogenesis and lymphangiogenesis have been recognized as critical processes in tumour growth and metastasis development, and numerous studies have evaluated the significance of these parameters in predicting the prognosis in solid tumours, including MM. We set out to determine whether angiogenesis, lymphangiogenesis and lymphatic invasion (LI) are valuable prognostic markers in MM. We systematically reviewed the available literature and subsequently performed a meta-analysis on the compiled data. To be eligible for the systematic review, a study had to provide the microvessel density (MVD), the lymphatic vessel density (LVD) or information about LI, assessed by immunohistochemistry on the primary site in patients with MM. To be evaluable for the meta-analysis, a study also had to provide information on clinical outcome. We approached selected studies with the Reporting recommendations for tumour marker (REMARK) criteria, verifying whether they had followed the recommendations. In total, nine angiogenesis, seven lymphangiogenesis and 10 LI studies were included in our meta-analysis, representing 419, 474 and 802 patients, respectively. Using meta-analysis, we showed that peritumoral LVD and the presence of LI have prognostic value for patients with MM. In contrast, MVD and intratumoral LVD did not have prognostic value in these patients. LVD and LI seem to have prognostic value for patients with MM.

Angiogenesis and lymphangiogenesis are complex processes, driven by multiple factors. In primary breast tumours (PTs), VEGFA, -C and -D are the most important (lymph)angiogenic factors. The induction of lymphangiogenesis in axillary lymph node (LN) metastases of patients with breast cancer was described recently. To compare the molecular determinants of (lymph)angiogenesis in LN metastases and PTs of breast cancer patients, RNA was isolated from formalin-fixed, paraffin-embedded tissue sections of a metastatically involved and uninvolved LN and the PT from 26 lymph node-positive patients. The expression of 12 (lymph)angiogenic markers was measured by qRT-PCR. Expression was correlated with tumour cell proliferation, angiogenesis and lymphangiogenesis, quantified by tumour cell proliferation fraction (TCP%) and (lymphatic) endothelial cell proliferation fraction [(L)ECP%]. TCP%, ECP% and LECP% were assessed on immunohistochemical double stains for CD34/Ki-67 and D2-40/Ki-67, respectively. In involved LNs, the relative gene expression levels of PROX1 (p < 0.001) and FGF2 (p = 0.008) were decreased and the expression levels of VEGFA (p = 0.01) and PDGFB (p = 0.002) were increased compared to uninvolved LNs. The expression of most markers was increased in PTs compared to involved LNs. In metastatically involved LNs, the expression of VEGFA correlated with ECP% (r = 0.54, p = 0.009) and LECP% (r = 0.76, p < 0.001). In PTs, VEGFA correlated only with ECP% (r = 0.74, p < 0.001). VEGFD correlated with peritumoural LECP% (r = 0.61, p = 0.001) and with VEGFC (r = 0.78, p < 0.001). Linear regression analysis confirmed the expression of VEGFA as an independent predictor of ECP% in both PTs and LN metastases and of LECP% in LN metastases. The expression of VEGFD, but not of VEGFA, independently predicted peritumoural LECP% in PTs. Our results confirm existing data that, in PTs, angiogenesis and lymphangiogenesis are respectively driven by VEGFA and VEGFD. In contrast, in LN metastases, both processes seem to be driven by VEGFA. Lymphangiogenesis in PTs and in LN metastases might thus be driven by different factors.

Angiogenesis in canine mammary tumours (CMTs) has been described previously; however, only the intratumoural (IT) region has been studied and information on peritumoural (PT) angiogenesis is lacking. In this study, the blood vessel density (BVD), blood vessel perimeter (BVP) and blood vessel area (BVA) in IT and PT regions of 56 benign CMTs, 55 malignant CMTs and 13 samples of normal mammary gland tissue were analyzed. In addition, the blood endothelial cell proliferation (BECP) as an indicator of ongoing angiogenesis was investigated. The prognostic value of each parameter was also examined. Blood vessels and proliferating blood endothelial cells were present in IT and PT regions of both benign and malignant tumours. The vessels in the PT region had a significantly higher area and perimeter compared with those in the IT region. Malignant tumours showed significantly more vessels with a smaller total BVA and a higher BECP compared with benign tumours and control tissue. In the PT regions there was a significantly higher BVD, BVA and BVP compared with the vessels in control tissue. Only the IT and PT BVD and PT BECP in benign tumours allowed prediction of survival. The morphology of blood vessels in CMTs shows similarities with those in human breast cancer, which strengthens the case for the use of dogs with CMTs in comparative oncology trials.