Presentation of a PCR-nuclease protection strategy for minimal residual disease monitoring in B-ALL.
Methods for detecting residual malignant cells in patients suffering from lymphoid malignancies have neither been sufficiently sensitive nor easy to routinize, hampering a potential prediction of disease outcome. Taking advantage of clone-specific DNA sequences, generated during lymphocyte differentiation and the polymerase chain reaction, some strategies have been developed for several groups. Up to now the most specific and sensitive methodology, which consists of designing leukemia-specific oligonucleotides, requires sequencing of the complementary determining region III-DNA for each particular patient and is too laborious to be applied to each case for routine monitoring in most hospital laboratories. In an attempt to achieve an easy way to detect residual malignant cells in B lymphoproliferative diseases, we have used a new PCR-based approach, named here as PCR-nuclease protection assay, consisting of: (i) amplification of DNA segments corresponding to the complementarity determining region III of the immunoglobulin heavy chain genes from samples at disease diagnosis; (ii) isolation of the disease-specific single-stranded DNA; (iii) labeling of the single-stranded DNA to generate specific probes; (iv) hybridization to amplified DNA from samples corresponding to different disease phases; and (v) digestion with S1-nuclease. Using this approach, we could detect one malignant cell in a background of 10(5) healthy cells. The sensitivity and specificity of this approach compares with those of the above mentioned specific oligonucleotide strategy in detecting residual malignant B cells. Moreover, this strategy is much less tedious and could be used by most hospital laboratories.
Limón
A
A
Departament de Criobiologia i Teràpia Cel.lular, Hospital Duran i Reynals, Spain.
Bertran
J
J
Llucià
M
M
Carmona
M
M
Canals
C
C
Ortega
J J
JJ
García
J
J
Rueda
F
F
D001483:Base Sequence; D002051:Burkitt Lymphoma / Q000175:diagnosis*; D002648:Child; D017931:DNA Primers / Q000737:chemistry; D015326:Gene Rearrangement, B-Lymphocyte, Heavy Chain; D005819:Genetic Markers; D006801:Humans; D008969:Molecular Sequence Data; D018365:Neoplasm, Residual / Q000175:diagnosis*; D015345:Oligonucleotide Probes / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods; D015719:Single-Strand Specific DNA and RNA Endonucleases
D017931:DNA Primers; D005819:Genetic Markers; D015345:Oligonucleotide Probes; D015719:Single-Strand Specific DNA and RNA Endonucleases