Nucleic acid extraction from formalin-fixed paraffin-embedded cancer cell line samples: a trade off between quantity and quality?

BACKGROUND: Advanced genomic techniques such as Next-Generation-Sequencing (NGS) and gene expression profiling, including NanoString, are vital for the development of personalised medicines, as they enable molecular disease classification. This has become increasingly important in the treatment of cancer, aiding patient selection. However, it requires efficient nucleic acid extraction often from formalin-fixed paraffin-embedded tissue (FFPE). METHODS: Here we provide a comparison of several commercially available manual and automated methods for DNA and/or RNA extraction from FFPE cancer cell line samples from Qiagen, life Technologies and Promega. Differing extraction geometric mean yields were evaluated across each of the kits tested, assessing dual DNA/RNA extraction vs. specialised single extraction, manual silica column based extraction techniques vs. automated magnetic bead based methods along with a comparison of subsequent nucleic acid purity methods, providing a full evaluation of nucleic acids isolated. RESULTS: Out of the four RNA extraction kits evaluated the RNeasy FFPE kit, from Qiagen, gave superior geometric mean yields, whilst the Maxwell 16 automated method, from Promega, yielded the highest quality RNA by quantitative real time RT-PCR. Of the DNA extraction kits evaluated the PicoPure DNA kit, from Life Technologies, isolated 2-14× more DNA. A miniaturised qPCR assay was developed for DNA quantification and quality assessment. CONCLUSIONS: Careful consideration of an extraction kit is necessary dependent on quality or quantity of material required. Here we provide a flow diagram on the factors to consider when choosing an extraction kit as well as how to accurately quantify and QC the extracted material.

Isolation and antisense suppression of flavonoid 3', 5'-hydroxylase modifies flower pigments and colour in cyclamen.

BACKGROUND: Cyclamen is a popular and economically significant pot plant crop in several countries. Molecular breeding technologies provide opportunities to metabolically engineer the well-characterized flavonoid biosynthetic pathway for altered anthocyanin profile and hence the colour of the flower. Previously we reported on a genetic transformation system for cyclamen. Our aim in this study was to change pigment profiles and flower colours in cyclamen through the suppression of flavonoid 3', 5'-hydroxylase, an enzyme in the flavonoid pathway that plays a determining role in the colour of anthocyanin pigments. RESULTS: A full-length cDNA putatively identified as a F3'5'H (CpF3'5'H) was isolated from cyclamen flower tissue. Amino acid and phylogeny analyses indicated the CpF3'5'H encodes a F3'5'H enzyme. Two cultivars of minicyclamen were transformed via Agrobacterium tumefaciens with an antisense CpF3'5'H construct. Flowers of the transgenic lines showed modified colour and this correlated positively with the loss of endogenous F3'5'H transcript. Changes in observed colour were confirmed by colorimeter measurements, with an overall loss in intensity of colour (C) in the transgenic lines and a shift in hue from purple to red/pink in one cultivar. HPLC analysis showed that delphinidin-derived pigment levels were reduced in transgenic lines relative to control lines while the percentage of cyanidin-derived pigments increased. Total anthocyanin concentration was reduced up to 80% in some transgenic lines and a smaller increase in flavonol concentration was recorded. Differences were also seen in the ratio of flavonol types that accumulated. CONCLUSION: To our knowledge this is the first report of genetic modification of the anthocyanin pathway in the commercially important species cyclamen. The effects of suppressing a key enzyme, F3'5'H, were wide ranging, extending from anthocyanins to other branches of the flavonoid pathway. The results illustrate the complexity involved in modifying a biosynthetic pathway with multiple branch points to different end products and provides important information for future flower colour modification experiments in cyclamen.

Vandetanib inhibits both VEGFR-2 and EGFR signalling at clinically relevant drug levels in preclinical models of human cancer.

Vandetanib is a multi-targeted receptor tyrosine kinase inhibitor that is in clinical development for the treatment of solid tumours. This preclinical study examined the inhibition of two key signalling pathways (VEGFR-2, EGFR) at drug concentrations similar to those achieved in the clinic, and their contribution to direct and indirect antitumour effects of vandetanib. For in vitro studies, receptor phosphorylation was assessed by Western blotting and ELISA, cell proliferation was assessed using a cell viability endpoint, and effects on cell cycle determined using flow cytometry. For in vivo studies, Western blotting, ELISA and immunohistochemistry (IHC) were used to assess receptor phosphorylation. Cell culture experiments demonstrated that anti-proliferative effects of vandetanib resulted from inhibition of either EGFR or VEGFR-2 signalling in endothelial cells, but were associated with inhibition of EGFR signalling in tumour cells. Vandetanib inhibited both EGFR and VEGFR-2 signalling in normal lung tissue and in tumour xenografts. In a lung cancer model expressing an activating EGFR mutation, the activity of vandetanib was similar to that of a highly selective EGFR inhibitor (gefitinib), and markedly greater than that of a highly selective VEGFR inhibitor (vatalanib). These data suggest that at the plasma exposures achieved in the clinic, vandetanib will significantly inhibit both VEGFR-2 and EGFR signalling, and that both inhibition of angiogenesis and direct inhibition of tumour cell growth can contribute to treatment response.

Identification of biomarkers in human head and neck tumor cell lines that predict for in vitro sensitivity to gefitinib.

Potential biomarkers were identified for in vitro sensitivity to the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor gefitinib in head and neck cancer. Gefitinib sensitivity was determined in cell lines, followed by transcript profiling coupled with a novel pathway analysis approach. Eleven cell lines were highly sensitive to gefitinib (inhibitor concentration required to give 50% growth inhibition [GI(50)] < 1 microM), three had intermediate sensitivity (GI(50) 1-7 microM), and six were resistant (GI(50) > 7 microM); an exploratory principal component analysis revealed a separation between the genomic profiles of sensitive and resistant cell lines. Subsequently, a hypothesis-driven analysis of Affymetrix data (Affymetrix, Inc., Santa Clara, CA, USA) revealed higher mRNA levels for E-cadherin (CDH1); transforming growth factor, alpha (TGF-alpha); amphiregulin (AREG); FLJ22662; EGFR; p21-activated kinase 6 (PAK6); glutathione S-transferase Pi (GSTP1); and ATP-binding cassette, subfamily C, member 5 (ABCC5) in sensitive versus resistant cell lines. A hypothesis-free analysis identified 46 gene transcripts that were strongly differentiated, seven of which had a known association with EGFR and head and neck cancer (human EGF receptor 3 [HER3], TGF-alpha, CDH1, EGFR, keratin 16 [KRT16], fibroblast growth factor 2 [FGF2], and cortactin [CTTN]). Polymerase chain reaction (PCR) and enzyme-linked immunoabsorbant assay analysis confirmed Affymetrix data, and EGFR gene mutation, amplification, and genomic gain correlated strongly with gefitinib sensitivity. We identified biomarkers that predict for in vitro responsiveness to gefitinib, seven of which have known association with EGFR and head and neck cancer. These in vitro predictive biomarkers may have potential utility in the clinic and warrant further investigation.