High Resolution Melting Analysis in Cancer Diagnosis

Abstract

The request of rapid and reliable screenings of somatic variants in human cancers is rapidly increasing to better define clinical samples and orient targeted therapies. The primary technical challenge encountered in the detection of somatic variants is the cellular heterogeneity in cancer biopsies. Genetic variants can be present in low amount within an elevated background of wild type sequences and more sensitive assays are therefore needed than those used for germline variants. High resolution melting analysis(HRM) was proposed as a very sensitive scanning method that allows rapid detection of DNA sequence variations, without cumbersome post-PCR procedures. Mutation scanning with HRM is based on the dissociation behaviour of DNA when exposed to increasing temperatures. The signal modification is generated from the transition from double-to-single strand in the presence of fluorescent dyes actively intercalating double-stranded DNA. The HRM profile gives a specific sequence-related pattern which differentiates wild type sequences from homo-/heterozygote variants. Co-amplification at lower denaturation temperature PCR (COLD-PCR) is a recently introduced method that allows preferential amplification of minority alleles from a mixture of wild type and mutant sequences. Preferential amplification of minor alleles is achieved by a change in denaturating temperature that characterizes each sequence with a nucleotide mismatch. COLD-PCR can identify mutated samples not otherwise identified by conventional PCR and cycle sequencing. COLD-PCR combined with HRM permits the correct identification of less represented mutations in cancer samples and better selection of patients eligible for targeted therapies, without requiring expensive and time-consuming procedures. HRM COLD-PCR has the potential to improve the routine search for mutations in cancer tissues. HRMA was also proposed as a rapid and sensitive technique for the assessment of DNA methylation. Recently we developed an optimized procedure, based on differential high resolution melting analysis (D-HRM), for the rapid and accurate quantification of methylated DNA. In D-HRMA two sets of primers are used in a single tube for the simultaneous amplification of the methylated and unmethylated DNA sequences. After HRM, differential fluorescence was calculated at the specific Tm after automatic subtraction of unmethylated-DNA fluorescence. Quantification was calculated by interpolation on an external standard curve generated with serial dilution of methylated-DNA.