Semiquantitative polymerase chain reaction for t(14;18) in follicular lymphomas: a colorimetric approach.
BACKGROUND
Autologous bone marrow transplantation is increasingly being used in the management of several types of cancer, and to avoid reintroduction of malignant cells, bone marrow purging is often performed. In such cases, sensitive quantitation methods are needed both to assess the efficacy of the purging and for surveillance of patients in remission. Polymerase chain reaction (PCR) has the necessary sensitivity for this application, but it requires that the cancer cells can be recognized by a defined genetic abnormality. In addition, PCR is in principle a qualitative technique and must be modified for quantitative purposes. In follicular non-Hodgkin's lymphomas, the translocation t(14;18) (q32;q21) is common and is used here for model experiments.


METHODS
A PCR-based method for the quantitation of translocation-positive cells was developed on the basis of coamplification of cancer-specific target molecules with competitor molecules of known concentration. Gel electrophoresis was substituted by a colorimetric quantitation system to cope with patient PCR products of the same size as the competitor product and ease automation of the method at a later stage. Cell line Karpas 422, which contains the t(14;18) (q32;21) translocation, was used to validate the method. The method was used to assess the efficacy of a patient bone marrow purging where the initial infiltration levels were too low for traditional detection systems.


RESULTS
A reproducible and near linear response was obtained between 70 pg and 200 ng K422 DNA, equivalent to 10 K422 cells and 30,000 K422 cells, respectively. Bone marrow infiltration in one patient was 0.6 to 0.7% before malignant cell removal and 3 to 7 x 10(-4) after removal. The corresponding figures for the other patient were 2% and 3 to 7 x 10(-4), respectively.


CONCLUSIONS
The method presented has a sufficient dynamic range for applications like evaluation of bone marrow purging or monitoring of minimal residual disease. Adaptation of this method to other translocations is discussed.
Deggerdal
A H
AH
Department of Immunology, Norwegian Radium Hospital, Oslo.
Pettersen
F
F
Kvalheim
G
G
Hornes
E
E
Smeland
E
E
Fodstad
O
O
Hovig
E
E
D001483:Base Sequence; D016465:Bone Marrow Purging; D002883:Chromosomes, Human, Pair 14; D002887:Chromosomes, Human, Pair 18; D003124:Colorimetry; D004247:DNA / Q000235:genetics; D004586:Electrophoresis; D006801:Humans; D008224:Lymphoma, Follicular / Q000235:genetics* / Q000473:pathology; D015335:Molecular Probes / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D011159:Population Surveillance; D012074:Remission Induction; D014178:Translocation, Genetic
D015335:Molecular Probes; D004247:DNA