Multilaboratory Assessment of a New Reference Material for Quality Assurance of Cell-Free Tumor DNA Measurements.

We conducted a multilaboratory assessment to determine the suitability of a new commercially available reference material with 40 cancer variants in a background of wild-type DNA at four different variant allele frequencies (VAFs): 2%, 0.50%, 0.125%, and 0%. The variants include single nucleotides, insertions, deletions, and two structural variations selected for their clinical importance and to challenge the performance of next-generation sequencing (NGS) methods. Fragmented DNA was formulated to simulate the size distribution of circulating wild-type and tumor DNA in a synthetic plasma matrix. DNA was extracted from these samples and characterized with different methods and multiple laboratories. The various extraction methods had differences in yield, perhaps because of differences in chemistry. Digital PCR assays were used to measure VAFs to compare results from different NGS methods. Comparable VAFs were observed across the different NGS methods. This multilaboratory assessment demonstrates that the new reference material is an appropriate tool to determine the analytical parameters of different measurement methods and to ensure their quality assurance.

The role of AP-1, NF-kappaB and ROS/NOS in skin carcinogenesis: the JB6 model is predictive.

Generation of reactive oxygen species (ROS) stimulates transcription by activating transcription factors activator protein 1 (AP-1) and nuclear factor kappaB (NF-KB). The mouse epidermal JB6 cells constitute a model system that has significantly contributed to the understanding of these events. Clonal variants of JB6 cells are differentially responsive to transformation induced by tumor promoters such as phorbol esters (TPA), epidermal growth factor (EGF) and tumor necrosis factor alpha (TNF-alpha), as well as oxidative stress. TPA and EGF, acting through the MAP kinase pathway, activate AP-1 and subsequently NF-kappaB proteins and downstream transcription processes that are involved in the transformation response in transformation-sensitive (P+) JB6 cells. The effect of TNF-alpha is primarily on the NF-kappaB pathway. ROS and other free radicals can activate AP-1 and NF-KB transcription coordinately. In JB6 cells, both ERK/Fra-1 and NF-kappaB activity is essential for the transformation response. Inhibition of NF-kappaB and AP-1 activity abrogates transformation in JB6 cells as well as in transgenic mice and human keratinocytes. A similar effect is seen with antioxidants, which inhibit NF-kappaB and AP-1 activity as well as transformation in JB6 cells. The JB6 model is therefore valuable for monitoring early events in oxidative stress related signaling leading to carcinogenesis, and for identifying molecular targets for cancer chemoprevention.