Precast commercial polyacrylamide gels for separation of DNA amplificates by temperature gradient gel electrophoresis: application to clonality analysis of lymphomas.
The third complementary determining region (CDR-III) of the rearranged immunoglobulin heavy chain (IgH) genes represents a unique marker for a lymphocyte and its clonal descendants and can be amplified by the polymerase chain reaction (PCR) technique. This approach has markedly enhanced the sensitivity for detection of clonal lymphocyte populations in patients with malignant B-lymphoid neoplasias. To monitor minimal residual disease (MRD) in tissue specimens during or after antineoplastic treatment, the problem of detecting the presence of a few clonal (malignant) lymphocytes in coexistence with a majority of polyclonal lymphocytes has to be addressed. Semi-nested PCR amplification of CDR-III rearrangements from specimen infiltrated by tumor cells generates clonal signals in front of a polyclonal background, and therefore high resolution electrophoretic techniques for separation of DNA fragments are required. Temperature gradient gel electrophoresis (TGGE) resolving DNA homo- and heteroduplexes according to their thermal stability has been successfully applied for this purpose using special electrophoretic equipment. We describe an adjustment to this technique by using a commercially available precast 0.5 mm thick polyacrylamide gel and by changing a standard horizontal electrophoretic device into a TGGE device. By this means we screened patients with B-cell lymphoma undergoing high-dosage radiochemotherapy followed by autologous transplantation for continuous presence of clonal (tumor-specific) CDR-III rearrangements. Specimens from blood and bone marrow were collected on diagnosis as well as before and after autologous transplantation. In addition, the autograft (bone marrow or peripheral blood hematopoietic stem cells) was analyzed. Tumor cells were easily detected in the transplants and in specimens collected during follow-up examinations. The clinical value of these findings remains unclear as yet because the number of cases investigated was small and the follow-up time is still too short. However, we conclude that the technique of combining the sensitivity of PCR with the specificity of high resolution TGGE is easy to use, making it possible to handle, in a clinical routine, a great number of samples within a short time in order to monitor MRD in patients with B-cell neoplasias.
Suttorp
M
M
Department of Pediatrics, University of Kiel, Germany.
von Neuhoff
N
N
Tiemann
M
M
Dreger
P
P
Schaub
J
J
Löffer
H
H
Parwaresch
R
R
Schmitz
N
N
D017690:Cell Transplantation; D004273:DNA, Neoplasm / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006412:Hematopoietic Stem Cells; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D008214:Lymphocytes / Q000502:physiology; D016393:Lymphoma, B-Cell / Q000097:blood / Q000175:diagnosis / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011933:Reagent Kits, Diagnostic; D013696:Temperature
D004273:DNA, Neoplasm; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D011933:Reagent Kits, Diagnostic