Analysis of tumor-specific gene rearrangements in pre-pre-B-ALL human BM biopsies engrafted in SCID mice. During lymphoid development, exons encoding TcR and Ig are assembled by a site-specific V(D)J recombination. The joining of coding ends is imprecise, and base loss or addition occurs. As a consequence, PCR amplification of TcR or IgH loci of polyclonal lymphocytes yields products of heterogenous size, reflecting the diversity of the N-region. This can be evidenced by size separation using polyacrylamide gel electrophoresis, which shows a gaussian distribution of PCR products according to size. Conversely, electrophoresis of amplification products obtained from a monoclonal population of lymphoid cells with a given rearranged locus results in a single peak. This latter observation is consistent with the presence of a unique N-region in all lymphocytes, and is thus characteristic of a malignant clone. The size of the rearrangement is highly clone-specific. To look for the presence of blastic cells in BM biopsies, mononucleated cell DNA from tissues before and after engraftment was amplified using fluorescein-labeled oligonucleotides and submitted to high-resolution polyacrylamide electrophoresis on a fluorescent automated laser DNA sequencer. PCR products were detected by fluorescence and visualized as peaks separated according to size. (A) Blast cells from sample R41 engrafted for 11 weeks contain a clonal IgH rearrangement on both alleles (b), identical to that detected in the biopsy before implantation (a). Similarly, R49 blast cells exhibit the same single-allele IgH rearrangement before (e) and after (d) a 6-week implantation in a SCID mouse. As a control, polyclonal IgH rearrangements present in a normal BM cell population were used (c). (B) The same Vγ9-J1J2 single-allele rearrangement is detected in R44 blasts analyzed at first diagnosis (b) and after transplantation of the BM biopsy in a SCID mouse for 5 weeks (a). Y-axis, intensity of fluorescence; X-axis, time (min).