BRAFV600E is a unique molecular marker for metastatic melanoma, being the most frequent somatic point mutation in this malignancy. Detection of BRAFV600E in blood could have prognostic and predictive value and could be useful for monitoring response to BRAF-targeted therapy. We developed a rapid, sensitive method for the detection and quantification of BRAFV600E in circulating free DNA (cfDNA) isolated from plasma and serum on the basis of a quantitative 5'-nuclease PCR (Taqman) in the presence of a peptide-nucleic acid. We validated the assay in 92 lung, colon, and melanoma archival serum and plasma samples with paired tumor tissue (40 wild-type and 52 BRAFV600E). The correlation of cfDNA BRAFV600E with clinical parameters was further explored in 22 metastatic melanoma patients treated with BRAF inhibitors. Our assay could detect and quantify BRAFV600E in mixed samples with as little as 0.005% mutant DNA (copy number ratio 1 : 20 000), with a specificity of 100% and a sensitivity of 57.7% in archival serum and plasma samples. In 22 melanoma patients treated with BRAF inhibitors, the median progression-free survival was 3.6 months for those showing BRAFV600E in pretreatment cfDNA compared with 13.4 months for those in whom the mutation was not detected (P=0.021). Moreover, the median overall survival for positive versus negative BRAFV600E tests in pretreatment cfDNA differed significantly (7 vs. 21.8 months, P=0.017). This finding indicates that the sensitive detection and accurate quantification of low-abundance BRAFV600E alleles in cfDNA using our assay can be useful for predicting treatment outcome.

Five polymorphic restriction enzyme sites in the beta globin gene cluster (HindIII Gγ-Hind III Aγ-, Ava II β-and Hpa I and Bam HI 3'β-globin gene) were studied in individuals from 13 families: 13 homozygote patients for sickle cell anemia, two double heterozygotes (one SC and one S/β ), 35 AS heterozygotes (23 parents and 12 siblings), one father (A/β ), and three normal siblings. In addition, 17 normal unrelated Mexican subjects were studied. All subjects were from the state of Veracruz on the coast of the Gulf of Mexico. The Southern blot technique was used. Fifteen haplotypes were identified in the 142 chromosomes. Five were the most frequent: two haplotypes, (+-+++) (52.4%) and (--+-+) (19.0%) were associated with β chromosomes; two haplotypes, (--+++) (38.2%) and (---++) (19.7%), were linked with β chromosomes, and the fifth (--++-) was present in both types of chromosomes. Haplotype (+-+++) corresponded to the Bantu or Senegal type. With Hinc II analysis after PCR amplification in both the 5' and 3' regions of the ψβ-globin gene, it was possible to distinguish between these African types, as in the former both restriction sites are absent. This analysis was done in 23 β and 10 β subjects. All β chromosomes disclosed the Bantu type, while β were similar to Caucasians. Bantu and Benin haplotypes have been found with high frequency in African populations, indicating the great influence of African genes in the population of the Mexican coasts. In addition, two previously unidentified haplotypes were found: (++--+) and (-++++). These can be explainded by crossing-over events and/or by new mutations. © 1995 Wiley-Liss, Inc.

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