The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference.

These retrospective tumor biomarker analyses suggest a positive association between density of vascular endothelial cells (the predominant cell type expressing VEGF receptors) and tumor VEGF-A levels and magnitude of bevacizumab effect in ovarian cancer. The potential predictive value of MVD (CD31) and tumor VEGF-A is consistent with a mechanism of action driven by VEGF-A signaling blockade.

Despite all efforts made to develop predictive biomarkers for antiangiogenic therapies, no unambiguous markers have been identified so far. This is due to among others the lack of standardized tests. This study presents an improved microvessel density quantification method in tumor tissue based on stereological principles and using whole-slide images. Vessels in tissue sections of different cancer types were stained for CD31 by an automated and validated immunohistochemical staining method. The stained slides were digitized with a digital slide scanner. Systematic, uniform, random sampling of the regions of interest on the whole-slide images was performed semi-automatically with the previously published applications AutoTag and AutoSnap. Subsequently, an unbiased counting grid was combined with the images generated with these scripts. Up to six independent observers counted microvessels in up to four cancer types: colorectal carcinoma, glioblastoma multiforme, ovarian carcinoma and renal cell carcinoma. At first, inter-observer variability was found to be unacceptable. However, after a series of consensus training sessions and interim statistical analysis, counting rules were modified and inter-observer concordance improved considerably. Every CD31-positive object was counted, with exclusion of suspected CD31-positive monocytes, macrophages and tumor cells. Furthermore, if interconnected, stained objects were considered a single vessel. Ten regions of interest were sufficient for accurate microvessel density measurements. Intra-observer and inter-observer variability were low (intraclass correlation coefficient > 0.7) if the observers were adequately trained.

Prostate cancer is the second leading cause of cancer-associated death in men. Despite having a relatively lower tumor mutational burden than most tumor types, multiple gene fusions such as TMPRSS2:ERG have been characterized and linked to more aggressive disease. Individual tumor samples have been found to contain multiple fusions and it remains unknown whether these fusions increase tumor immunogenicity. Here, we investigated the role of fusion burden on the prevalence and expression of key molecular and immune effectors in prostate cancer tissue specimens which represented the different stages of disease progression and androgen sensitivity, including hormone sensitive and castration resistant prostate cancer. We found that tumor fusion burden was inversely correlated with tumor mutational burden and not associated with disease stage. High fusion burden correlated with high immune infiltration, PD-L1 expression on immune cells, and immune signatures representing activation of T cells and M1 macrophages.High fusion burden inversely correlated with immune suppressive signatures. Our findings suggest that high tumor fusion burden may be a more appropriate biomarker than tumor mutational burden in prostate cancer as it more closely associates with immunogenicity and suggests that tumors with high fusion burden could be potential candidates for immunotherapeutic agents.

Tumor angiogenesis is measured by counting microvessels in tissue sections at high power magnification as a potential prognostic or predictive biomarker. Until now, regions of interest1 (ROIs) were selected by manual operations within a tumor by using a systematic uniform random sampling2 (SURS) approach. Although SURS is the most reliable sampling method, it implies a high workload. However, SURS can be semi-automated and in this way contribute to the development of a validated quantification method for microvessel counting in the clinical setting. Here, we report a method to use semi-automated SURS for microvessel counting:Whole slide imaging with Pannoramic SCAN (3DHISTECH)Computer-assisted sampling in Pannoramic Viewer (3DHISTECH) extended by two self-written AutoHotkey applications (AutoTag and AutoSnap)The use of digital grids in Photoshop® and Bridge® (Adobe Systems)This rapid procedure allows traceability essential for high throughput protein analysis of immunohistochemically stained tissue.

Fibroblast activation protein-α (FAP) is a transmembrane peptidase and a surrogate marker for cancer-associated fibroblasts (CAFs). FAP has been linked to worse prognosis and therapy resistance in several cancers. We hypothesised that FAP might have a prognostic 3biomarker potential to stratify patients with high-grade (HG) T1 non-muscle-invasive bladder cancer (NMIBC). We selected 30 patients with HG T1 NMIBC that progressed to ≥T2 disease which were pair-matched based on CUETO progression score variables with 90 patients that did not progress. After revision a final cohort of 86 patients was retained. Slides were stained for FAP, the luminal marker GATA3 and the basal marker CK5. All HG T1 tumour regions of interest (ROIs) within each patient were annotated, analysed and scored using image analysis software. FAP expression in HG T1 ROIs was significantly higher in progressors vs. non-progressors and was prognostic for recurrence-free survival, progression-free survival, cancer-specific survival, and overall survival. FAP expression in HG T1 ROIs remained strongly prognostic for these outcomes in a bivariable model corrected for adequate BCG per FDA definition. Expression of GATA3 and CK5 did not differ between progressors vs. non-progressors, and were not prognostic for these outcomes. FAP might serve as an easily applicable prognostic biomarker to risk-stratify patients with HG T1 NMIBC if these results are prospectively validated in a larger series.

Bevacizumab is the first anti-angiogenic agent approved for the treatment of metastatic colorectal cancer. The need for patient selection before initiating therapy necessitates the study of various proteins expressed in metastatic colorectal cancer tissue as candidate predictive markers. Immunohistochemistry is a valuable, commonly available and cost-effective method to assess predictive biomarkers. However, it is subject to variations and therefore requires rigorous protocol standardizations. Furthermore, validated quantification methodologies to study these angiogenic elements have to be applied. Based on their function in tumor angiogenesis and their relation to the mechanism of action of bevacizumab, protein markers were divided in four groups: VEGF A-signaling proteins; other relevant angiogenesis factors; factors regarding the tumor microenvironment and tumor intrinsic markers. Conceivably, nimbly selecting a small but relevant group of therapy-guided patients by the appropriate combination of predictive biomarkers may confer great value to this angiogenic inhibitor.

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