Establishment and study of different real-time polymerase chain reaction assays for the quantification of cells with deletions of chromosome 7.
The evaluation of residual disease, which has prognostic value in the treatment of hematological malignancies, is currently assessed by scoring a limited number of cells by karyotyping and molecular cytogenetics. Real-time polymerase chain reaction (PCR) is an easier and more sensitive technique, enables analysis of a larger number of cells, and decreases sampling error. However, real-time PCR has been applied only to target transcripts of fusion genes. Here, we considered two real-time PCR strategies to quantify a number of cells carrying a partial deletion of chromosome 7 mixed with normal disomic cells. The first strategy was based on the amplification of two sequences, one on chromosome 7 and the other on chromosome 14. In the second strategy residual disease was assessed by the ratio between the two alleles of a bi-allelic marker, mapped on chromosome 7, measured with allele-specific assays. Precision and accuracy of the two approaches were tested by reference samples with nominal values of residual disease ranging from 2 to 95%. As expected the second strategy resulted in more precise and accurate monitoring within the range from 5 to 95%. Furthermore, this method may be applied to assess the number of dysplastic or neoplastic clones carrying any unbalanced chromosome changes.
Mattarucchi
Elia
E
Department of Experimental and Clinical Biomedical Sciences, University of Insubria, Via Dunant 5, 21100 Varese, Italy.
Marsoni
Milena
M
Passi
Alberto
A
Lo Curto
Francesco
F
Pasquali
Francesco
F
Porta
Giovanni
G
D001483:Base Sequence; D002872:Chromosome Deletion; D002897:Chromosomes, Human, Pair 7 / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D013997:Time Factors