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

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 KRAS (32%), TP53 (32%), BRAF (12%), APC (11%), EGFR (8%) and NRAS (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.

Prognostic role of vasculogenic mimicry in colorectal cancer.

PURPOSE: Angiogenesis, as measured by degree of microvessel density, has been associated with tumor progression in many tumor types but does not always correlate with clinical outcome. In 1999, aggressive tumor cells were shown to form blood-conducting tubes not lined by endothelial cells, and this phenomenon was termed vasculogenic mimicry. We investigated angiogenesis and the presence of vasculogenic mimicry in colorectal carcinoma in relation to tumor stage, patient survival, and genetic indicators of tumor cell plasticity. METHODS: Paraffin-embedded tissue samples were examined from a study of 117 patients with colorectal carcinoma with a 12-year follow-up. Immunohistochemical analysis was used to measure microvessel density and proliferating endothelial cells and to detect vasculogenic mimicry (scored by 3 independent observers). Cell cultures from 7 colon cell lines, real-time polymerase chain reaction (PCR) on cell lines, frozen tissue material from 4 colorectal cancer patients with and 4 without vasculogenic mimicry, and fresh colorectal cancer tissue from 2 patients were used to investigate the relationship between vasculogenic mimicry and tumor cell plasticity. RESULTS: Microvessel density was not a prognostic marker in our patients. We found vasculogenic mimicry in 23 (19.7%) of 117 colorectal tumor samples. Cell culture experiments and real-time PCR on human colorectal carcinoma material showed evidence for vasculogenic mimicry with overexpression of EPHA2 and LAMC2, known to be important for the tube-forming capacity of melanoma tumor cells. The presence of vasculogenic mimicry was associated with significantly shortened survival, both overall (P < 0.0001) and within intermediate cancer stages (Dukes B, P = 0.0277; Dukes C, P < 0.0001). CONCLUSIONS: Vasculogenic mimicry can occur in colorectal carcinoma and appears to be comparable to vasculogenic mimicry described in other tumors. Moreover, vasculogenic mimicry in colorectal carcinoma may be a strong independent prognostic marker for survival.

A transgenic Tie2-GFP athymic mouse model; a tool for vascular biology in xenograft tumors.

We report the generation of a transgenic Tie2-GFP athymic nude mouse, carrying green fluorescent blood vessels throughout the body. This transgenic mouse is a tool for studies in vascular biology, and is especially of interest for imaging of tumor angiogenesis and the study of anti-angiogenesis strategies in (human) xenografts. Intravital microscopy identified the presence of blood conducting structures that are not lined by endothelial cells. Dedifferentiation of aggressive tumor cells can lead to acquisition of endothelial characteristics. This process of tumor cell plasticity, also referred to as vasculogenic mimicry, has been suggested to contribute to the circulatory system in a tumor. In plastic EW7 Ewing sarcoma tumors in these Tie2-GFP mice, we observed blood flow in both regular blood vessels and non-fluorescent tumor cell-lined channels, visualizing in vivo hemodynamics in vasculogenic channels. These results demonstrate that the transgenic Tie2-GFP athymic mouse model is a valuable tool for vascular biology research.

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.

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.

Effects of radiotherapy and chemotherapy on angiogenesis and leukocyte infiltration in rectal cancer.

BACKGROUND: We and others have shown that angiogenesis and leukocyte infiltration are important prognostic factors in rectal cancer. However, little is known about its possible changes in response to radiotherapy (RTX), which is frequently given to rectal tumors as a neoadjuvant treatment to improve the prognosis. We therefore investigated the biologic effects of RTX on these parameters using fresh-frozen biopsy samples of tumor and normal mucosa tissue before and after RTX. METHODS: Biopsy samples were taken from a total of 34 patients before and after either a short course or long course of RTX combined with chemotherapy. The following parameters were analyzed by immunohistochemistry, flow cytometry, or quantitative real-time polymerase chain reaction: Microvessel density, leukocyte infiltration, proliferating epithelial and tumor cells, proliferating endothelial cells, adhesion molecule expression on endothelial cells, and the angiogenic mRNA profile. RESULTS: The tumor biopsy samples taken after RTX treatment demonstrated a significant decrease in microvessel density and the number of proliferating tumor cells and proliferating endothelial cells (p < 0.001). In contrast, the leukocyte infiltration, the levels of basic fibroblast growth factor in carcinoma tissue, and the adhesion molecule expression on endothelial cells in normal as well as carcinoma tissue increased significantly (p < 0.05). CONCLUSION: Our data show that together with an overall decrease in tumor cell and endothelial cell proliferation, RTX results in an increase in the expression of adhesion molecules that stimulate leukocyte infiltration. This suggests the possibility that, in addition to its direct cytotoxic effect, radiation may also stimulate an immunologic tumor response that could contribute to the documented improvement in local tumor control and distal failure rate of rectal cancers.

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.

Tumor cell plasticity in Ewing sarcoma, an alternative circulatory system stimulated by hypoxia.

A striking feature of Ewing sarcoma is the presence of blood lakes lined by tumor cells. The significance of these structures, if any, is unknown. Here, we report that the extent of blood lakes correlates with poor clinical outcomes, whereas variables of angiogenesis do not. We also show that Ewing sarcoma cells form vessel-like tubes in vitro and express genes associated with vasculogenic mimicry. In tumor models, we show that there is blood flow through the blood lakes, suggesting that these structures in Ewing sarcoma contribute to the circulation. Furthermore, we present evidence that reduced oxygen tension may be instrumental in tube formation by plastic tumor cells. The abundant presence of these vasculogenic structures, in contrast to other tumor types, makes Ewing sarcoma the ideal model system to study these phenomena. The results suggest that optimal tumor treatment may require targeting of these structures in combination with prevention of angiogenesis.