title: Real-time t(11;14) and t(14;18) PCR assays provide sensitive and quantitative assessments of minimal residual disease (MRD).
abstract: Non-Hodgkin's lymphoma (NHL) arises as a clonal transformation of normal B and T cell differentiation and is often characterized by a higher incidence of specific chromosomal translocations. We have developed real-time TaqMan PCR assays directed toward two of these tumor-associated DNA markers, the t(14;18)(q32;q21.3) at the major breakpoint region of the bcl-2 gene and the t(11;14)(q13;q32) at the bcl-1 major translocation cluster. During analysis of serial dilutions of t(14;18)-positive DNA, the t(14;18) real-time PCR was at least as sensitive as nested PCR and demonstrated enhanced quantitative potential. Moreover, in a blinded comparison of the t(14;18) real-time PCR and a clinically validated nested PCR protocol using 134 cell line and patient DNA samples, the real-time PCR detected the translocation in 30.0% more cases than nested PCR. Both the t(14;18) and t(11;14) real-time PCR assays were used to quantitate minimal residual disease (MRD) in an NHL clinical trial assessing the safety and efficacy of a tumor-purging protocol in autologous stem cell transplantation. The assays were also used to evaluate disease depletion in an ex vivo tumor spiking model in which normal peripheral blood was spiked with tumor cell lines and processed according to the clinical purging method. PCR data from both the clinical trial and the ex vivo model demonstrated a 4 to 6 log reduction in tumor cells during CD34+ and CD34+ Thy-1+ enrichment. Because the t(14;18) and t(11;14) real-time PCR assays are very sensitive, quantitative, rapid, and require no post-PCR manipulation, they may serve as practical alternatives to nested PCR.
authors.affiliation: SyStemix Inc., A Novartis Company, Palo Alto, CA, USA.
authors.forename: K
authors.initials: K
authors.lastname: Olsson
authors.forename: C J
authors.initials: CJ
authors.lastname: Gerard
authors.forename: J
authors.initials: J
authors.lastname: Zehnder
authors.forename: C
authors.initials: C
authors.lastname: Jones
authors.forename: R
authors.initials: R
authors.lastname: Ramanathan
authors.forename: C
authors.initials: C
authors.lastname: Reading
authors.forename: E G
authors.initials: EG
authors.lastname: Hanania
mesh_terms: D018952:Antigens, CD34 / Q000032:analysis; D001483:Base Sequence; D001854:Bone Marrow Cells / Q000378:metabolism; D016465:Bone Marrow Purging; D019457:Chromosome Breakage / Q000235:genetics; D002880:Chromosomes, Human, Pair 11 / Q000235:genetics; D002883:Chromosomes, Human, Pair 14 / Q000235:genetics; D002887:Chromosomes, Human, Pair 18 / Q000235:genetics; D002986:Clinical Trials as Topic; D019940:Genes, bcl-1 / Q000235:genetics; D019254:Genes, bcl-2 / Q000235:genetics; D006801:Humans; D008228:Lymphoma, Non-Hodgkin / Q000235:genetics* / Q000378:metabolism / Q000473:pathology* / Q000628:therapy; D008969:Molecular Sequence Data; D018365:Neoplasm, Residual; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D016037:Single-Blind Method; D018800:Thy-1 Antigens / Q000032:analysis; D013997:Time Factors; D014178:Translocation, Genetic / Q000235:genetics*; D014407:Tumor Cells, Cultured
chemical_list: D018952:Antigens, CD34; D018800:Thy-1 Antigens