Detection of somatic mutations by high-resolution DNA melting (HRM) analysis in multiple cancers.
Identification of somatic mutations in cancer is a major goal for understanding and monitoring the events related to cancer initiation and progression. High resolution melting (HRM) curve analysis represents a fast, post-PCR high-throughput method for scanning somatic sequence alterations in target genes. The aim of this study was to assess the sensitivity and specificity of HRM analysis for tumor mutation screening in a range of tumor samples, which included 216 frozen pediatric small rounded blue-cell tumors as well as 180 paraffin-embedded tumors from breast, endometrial and ovarian cancers (60 of each). HRM analysis was performed in exons of the following candidate genes known to harbor established commonly observed mutations: PIK3CA, ERBB2, KRAS, TP53, EGFR, BRAF, GATA3, and FGFR3. Bi-directional sequencing analysis was used to determine the accuracy of the HRM analysis. For the 39 mutations observed in frozen samples, the sensitivity and specificity of HRM analysis were 97% and 87%, respectively. There were 67 mutation/variants in the paraffin-embedded samples, and the sensitivity and specificity for the HRM analysis were 88% and 80%, respectively. Paraffin-embedded samples require higher quantity of purified DNA for high performance. In summary, HRM analysis is a promising moderate-throughput screening test for mutations among known candidate genomic regions. Although the overall accuracy appears to be better in frozen specimens, somatic alterations were detected in DNA extracted from paraffin-embedded samples.
Laboratory of Translational Genomics, Department of Health and Human Services, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America.
Jesus
J
Gonzalez-Bosquet
Jacob
J
Calcei
Jun S
JS
Wei
Montserrat
M
Garcia-Closas
Mark E
ME
Sherman
Stephen
S
Hewitt
Joseph
J
Vockley
Jolanta
J
Lissowska
Hannah P
HP
Yang
Javed
J
Khan
Stephen
S
Chanock
D001706:Biopsy; D004252:DNA Mutational Analysis / Q000379:methods; D004273:DNA, Neoplasm / Q000235:genetics*; D005629:Frozen Sections; D006801:Humans; D009154:Mutation; D009363:Neoplasm Proteins / Q000235:genetics; D009369:Neoplasms / Q000235:genetics*; D009691:Nucleic Acid Denaturation / Q000235:genetics*; D016612:Paraffin Embedding
D004273:DNA, Neoplasm; D009363:Neoplasm Proteins
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