A quantitative polymerase chain reaction-enzyme immunoassay for accurate measurements of human papillomavirus type 16 DNA levels in cervical scrapings

A quantitative polymerase chain reaction-enzyme immunoassay (Q-PCR-EIA) was developed to measure the amount of human papillomavirus (HPV) 16 DNA per genome equivalent in cervical scrapings. The quantitative approach was based on a combined competitive PCR for both HPV 16, using the general primer GP5+/6+ PCR, and β-globin DNA. The two competitive PCRs involve co-amplification of target sequences and exogenously added DNA constructs carrying a rearranged 30 bp sequence in the probe-binding region. The accuracy of quantification by combining the two competitive PCR assays was validated on mixtures of HPV 16 containing cervical cancer cells of CaSki and SiHa cell lines. Comparison of this fully quantitative PCR assay with two semi-quantitative HPV PCR assays on a series of crude cell suspensions from HPV 16 containing cervical scrapings revealed remarkable differences in the calculated relative HPV load between samples. We found evidence that correction for both intertube variations in PCR efficiency and number of input cells/integrity of DNA significantly influence the outcome of studies on viral DNA load in crude cell suspensions of cervical scrapings. Therefore, accurate measurements on viral DNA load in cervical scrapings require corrections for these phenomena, which can be achieved by application of this fully quantitative approach. © 1999 Cancer Research Campaig

Pan-cancer analysis of whole genomes

Cancer is driven by genetic change, and the advent of massively parallel sequencing has enabled systematic documentation of this variation at the whole-genome scale(1-3). Here we report the integrative analysis of 2,658 whole-cancer genomes and their matching normal tissues across 38 tumour types from the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA). We describe the generation of the PCAWG resource, facilitated by international data sharing using compute clouds. On average, cancer genomes contained 4-5 driver mutations when combining coding and non-coding genomic elements; however, in around 5% of cases no drivers were identified, suggesting that cancer driver discovery is not yet complete. Chromothripsis, in which many clustered structural variants arise in a single catastrophic event, is frequently an early event in tumour evolution; in acral melanoma, for example, these events precede most somatic point mutations and affect several cancer-associated genes simultaneously. Cancers with abnormal telomere maintenance often originate from tissues with low replicative activity and show several mechanisms of preventing telomere attrition to critical levels. Common and rare germline variants affect patterns of somatic mutation, including point mutations, structural variants and somatic retrotransposition. A collection of papers from the PCAWG Consortium describes non-coding mutations that drive cancer beyond those in the TERT promoter(4); identifies new signatures of mutational processes that cause base substitutions, small insertions and deletions and structural variation(5,6); analyses timings and patterns of tumour evolution(7); describes the diverse transcriptional consequences of somatic mutation on splicing, expression levels, fusion genes and promoter activity(8,9); and evaluates a range of more-specialized features of cancer genomes(8,10-18).Peer reviewe