Tumour vascularization: sprouting angiogenesis and beyond

Tumour angiogenesis is a fast growing domain in tumour biology. Many growth factors and mechanisms have been unravelled. For almost 30 years, the sprouting of new vessels out of existing ones was considered as an exclusive way of tumour vascularisation. However, over the last years several additional mechanisms have been identified. With the discovery of the contribution of intussusceptive angiogenesis, recruitment of endothelial progenitor cells, vessel co-option, vasculogenic mimicry and lymphangiogenesis to tumour growth, anti-tumour targeting strategies will be more complex than initially thought. This review highlights these processes and intervention as a potential application in cancer therapy. It is concluded that future anti-vascular therapies might be most beneficial when based on multimodal anti-angiogenic, anti-vasculogenic mimicry and anti-lymphangiogenic strategies

Proliferating endothelial cells, but not microvessel density, are a prognostic parameter in human cutaneous melanoma

The induction of angiogenesis is crucial in the development of most human tumors. Angiogenesis is routinely assessed by the density of tumor microvessels. This technique reveals controversial results on the clinical and prognostic value of angiogenesis in melanoma. We investigated angiogenesis in tumor tissues of 58 cutaneous melanoma patients, of which a clinical follow-up of over 10 years was available, through assessment of microvessel density and by enumeration of the number of proliferating endothelial cells. To that end, vessels were immunohistochemically detected by CD31/CD34 staining, and proliferating endothelial cells were enumerated in a double staining with the proliferation marker Ki67. We found that microvessel density did not correlate with tumor stage or survival, neither in intratumoral nor in peritumoral areas. In contrast, proliferating endothelial cells were only observed in intratumoral areas and were correlated positively with tumor stage and the presence of distant metastases. In addition, a strong positive correlation was found with the number of proliferating tumor cells. Finally, high numbers of growing endothelial cells predicted short survival. Our results show that angiogenesis could best be measured by enumeration of proliferating endothelial cells and that this parameter has prognostic value in patients with cutaneous melanoma

Leukocyte infiltration and tumor cell plasticity are parameters of aggressiveness in primary cutaneous melanoma

Various clinical and experimental observations detected an immunological host defense in cutaneous melanoma. In order to investigate the prognostic value of leukocyte effector mechanisms, we examined the presence of different subsets of leukocytes in tumor samples of 58 patients diagnosed with primary cutaneous melanoma. The presence of T lymphocytes, cytotoxic T lymphocytes, B lymphocytes, CD16+ cells and macrophages was correlated to Breslow depth. A significantly higher amount of several subsets of leukocytes was found in samples with a more progressed tumor stage and survival analysis demonstrated that a higher amount of T lymphocytes and CD16+ cells was associated with a short survival. The amount of FOXP3+ regulatory T lymphocytes did not correlate with survival, nevertheless, it correlated with the amount of total infiltrate. In contrast, analysis of the expression of CD69, a marker for activated lymphocytes, demonstrated that patients with a higher amount of CD69+ lymphocytes had a better survival. In addition, a new parameter for aggressiveness of melanoma, tumor cell plasticity [i.e., the presence of periodic acid Schiff's (PAS) reagent positive loops], also predicted short survival and a trend of a higher amount of tumor infiltrating leukocytes in tumors with PAS positive loops was observed. These findings demonstrate that leukocyte infiltration and the presence of PAS loops is a sign of tumor aggressiveness and may have prognostic value

Validation and Application of a Custom-Designed Targeted Next-Generation Sequencing Panel for the Diagnostic Mutational Profiling of Solid Tumors

<div><p>The inevitable switch from standard molecular methods to next-generation sequencing for the molecular profiling of tumors is challenging for most diagnostic laboratories. However, fixed validation criteria for diagnostic accreditation are not in place because of the great variability in methods and aims. Here, we describe the validation of a custom panel of hotspots in 24 genes for the detection of somatic mutations in non-small cell lung carcinoma, colorectal carcinoma and malignant melanoma starting from FFPE sections, using 14, 36 and 5 cases, respectively. The targeted hotspots were selected for their present or future clinical relevance in solid tumor types. The target regions were enriched with the TruSeq approach starting from limited amounts of DNA. Cost effective sequencing of 12 pooled libraries was done using a micro flow cell on the MiSeq and subsequent data analysis with MiSeqReporter and VariantStudio. The entire workflow was diagnostically validated showing a robust performance with maximal sensitivity and specificity using as thresholds a variant allele frequency >5% and a minimal amplicon coverage of 300. We implemented this method through the analysis of 150 routine diagnostic samples and identified clinically relevant mutations in 16 genes including <i>KRAS</i> (32%), <i>TP53</i> (32%), <i>BRAF</i> (12%), <i>APC</i> (11%), <i>EGFR</i> (8%) and <i>NRAS</i> (5%). Importantly, the highest success rate was obtained when using also the low quality DNA samples. In conclusion, we provide a workflow for the validation of targeted NGS by a custom-designed pan-solid tumor panel in a molecular diagnostic lab and demonstrate its robustness in a clinical setting.</p></div

Summary of the validation assays and results for the targeted NGS screening on solid tumors (NSCLC, CRC and MELA).

<p>Summary of the validation assays and results for the targeted NGS screening on solid tumors (NSCLC, CRC and MELA).</p

Limit-of-detection assay.

<p>Two samples with known mutations were mixed at different percentages, captured and sequenced. VAFs of each mutation were plotted against the percentage in the mix. (A) Mutations KRAS G13D (58%), FBXW7 R505S 48%), APC K1462fs (52%), NRAS Q61R (35%) with high VAFs in the original sample. (B) Mutations NRAS Q61L (9%), ERBB2 V842I (8%), TP53 Y234* (9%), KRAS D12D (24%) with low VAFs in the original sample.</p

Flow chart for variant analysis.

<p>This chart is used for our classification of variants in solid tumor samples. VAF: Variant allele frequency; CDS: coding sequence; SNP: single nucleotide polymorphism; MAF: Minor allele frequency; IGV: Integrative Genomics Viewer; LoF: Loss-of-function.</p