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 p olymerase chain reaction (PCR) technique. This approach has markedly e nhanced 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 antineoplas tic treatment, the problem of detecting the presence of a few clonal ( malignant) lymphocytes in coexistence with a majority of polyclonal ly mphocytes has to be addressed. Semi-nested PCR amplification of CDR-II I rearrangements from specimen infiltrated by tumor cells generates cl onal signals in front of a polyclonal background, and therefore high r esolution electrophoretic techniques for separation of DNA fragments a re 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 electrophore tic 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 devi ce. 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 rearrangeme nts. Specimens from blood and bone marrow were collected on diagnosis as well as before and after autologous transplantation. In addition, t he autograft (bone marrow or peripheral blood hematopoietic stem cells ) was analyzed. Tumor cells were easily detected in the transplants an d in specimens collected during follow-up examinations. The clinical v alue of these findings remains unclear as yet because the number of ca ses investigated was small and the follow-up time is still too short. However, we conclude that the technique of combining the sensitivity o f PCR with the specificity of high resolution TGGE is easy to use, mak ing it possible to handle, in a clinical routine, a great number of sa mples within a short time in order to monitor MRD in patients with B-c ell neoplasias.