Background: The development of targeted therapies has created a pressing clinical need for the rapid and robust molecular characterisation of cancers. We describe here the application of highresolution melting analysis (HRM) to screen for KRAS mutations in clinical cancer samples. In nonsmall cell lung cancer, KRAS mutations have been shown to identify a group of patients that do not respond to EGFR targeted therapies and the identification of these mutations is thus clinically important.

Background: Epithelial growth factor receptor (EGFR) and KRAS mutation status have been reported as predictive markers of tumour response to EGFR inhibitors. High resolution melting (HRM) analysis is an attractive screening method for the detection of both known and unknown mutations as it is rapid to set up and inexpensive to operate. However, up to now it has not been fully validated for clinical samples when formalin-fixed paraffin-embedded (FFPE) sections are the only material available for analysis as is often the case.

DNA methylation changes that are recurrent in cancer have generated great interest as potential biomarkers for the early detection and monitoring of cancer. In such situations, essential information is missed if the methylation detection is purely qualitative. We describe a new probe-free quantitative methylation-specific PCR (MSP) assay that incorporates evaluation of the amplicon by high-resolution melting (HRM) analysis. Depending on amplicon design, different types of information can be obtained from the HRM analysis. Much of this information cannot be obtained by electrophoretic analysis. In particular, identification of false positives due to incomplete bisulphite conversion or false priming is possible. Heterogeneous methylation can also be distinguished from homogeneous methylation. As proof of principle, we have developed assays for the promoter regions of the CDH1, DAPK1, CDKN2A (p16INK4a) and RARB genes. We show that highly accurate quantification is possible in the range from 100% to 0.1% methylated template when 25 ng of bisulphite-modified DNA is used as a template for PCR. We have named this new approach to quantitative methylation detection, Sensitive Melting Analysis after Real Time (SMART)-MSP.

Introduction Individuals with germline mutations in the BRCA1 gene have an elevated risk of developing breast cancer, and often display characteristic clinicopathological features. We hypothesised that inactivation of BRCA1 by promoter methylation could occur as a germline or an early somatic event that predisposes to breast cancer with the phenotype normally associated with BRCA1 germline mutation.

Backgroundp53 is commonly inactivated by mutations in the DNA-binding domain in a wide range of cancers. As mutant p53 often influences response to therapy, effective and rapid methods to scan for mutations in TP53 are likely to be of clinical value. We therefore evaluated the use of high resolution melting (HRM) as a rapid mutation scanning tool for TP53 in tumour samples.MethodsWe designed PCR amplicons for HRM mutation scanning of TP53 exons 5 to 8 and tested them with DNA from cell lines hemizygous or homozygous for known mutations. We assessed the sensitivity of each PCR amplicon using dilutions of cell line DNA in normal wild-type DNA. We then performed a blinded assessment on ovarian tumour DNA samples that had been previously sequenced for mutations in TP53 to assess the sensitivity and positive predictive value of the HRM technique. We also performed HRM analysis on breast tumour DNA samples with unknown TP53 mutation status.ResultsOne cell line mutation was not readily observed when exon 5 was amplified. As exon 5 contained multiple melting domains, we divided the exon into two amplicons for further screening. Sequence changes were also introduced into some of the primers to improve the melting characteristics of the amplicon. Aberrant HRM curves indicative of TP53 mutations were observed for each of the samples in the ovarian tumour DNA panel. Comparison of the HRM results with the sequencing results revealed that each mutation was detected by HRM in the correct exon. For the breast tumour panel, we detected seven aberrant melt profiles by HRM and subsequent sequencing confirmed the presence of these and no other mutations in the predicted exons.ConclusionHRM is an effective technique for simple and rapid scanning of TP53 mutations that can markedly reduce the amount of sequencing required in mutational studies of TP53.

S500associated with HPV infection. Our recent report has shown that highrisk HPV 16/18 infections were associated with female lung cancer incidences in Taiwan. To address the correlation between the different p53 genotypes, HPV infection and HPV oncoprotein expression in p53 protein degradation of lung cancer, 60 health subjects and 141 primary lung cancer patients were included in this study. PCR-RFLP, nested-PCR and immunohistochemial analysis were used to detect the p53 codon 72 polymorphism, HPV 16/18 infection, HPV 16/18 E6 and p53 protein expression. Immunohistochemistry data showed that HPV 16 or 18 E6 protein was more frequently detected in female, adenocarcinoma, and nonsmoking lung tumors, compared with that of male, squamous cell carcinoma, and smoking lung tumors. Interestingly, HPV 16/18 E6 expressions but not HPV 16/18 DNA were reversely correlated with p53 expression, which was further confirmed by tissue in situ immunostaining. Additionally, the frequency of p53 protein degradation in HPV 16/18 E6-positive/Arg homozygosity lung cancer tumors is much higher than that in the other three categorizes. This correlation was not found in HPV DNA infection. In conclusion, HPV 16/18 E6 proteins involvement in p53 inactivation to contribute to HPV-infected lung tumorigenesis is correlate with p53 genotypes.