(A and D) DNA samples from patient cells were digested with restriction enzymes and ligated to linkers, after which integration site sequences were determined by PCR amplification and pyrosequencing. Numbers above bars denote total integration site reads, with composition broken down. PBL, peripheral blood lymphocytes. Longitudinal analysis of samples from P7 (A) and P10 (D). Time of chemotherapy is indicated by the arrow. (B and E) Analysis of genomic DNA samples by ligation-mediated PCR, separation by electrophoresis on an agarose gel, and staining with ethidium bromide. (B) In P7, the intense band in the M+68 sample had the mobility expected for the CCND2 amplification product. (E) In P10, bands of the expected sizes were observed for the LMO2 and BMI1 amplification products in the M+33 sample. (C and F) Longitudinal vector copy number analysis. Quantitative real-time PCR analysis of PBMC DNA from P7 and P10 obtained at the indicated times after gene therapy. (C) P7 showed 1 integrated copy in the leukemic blasts at M+68, concordant with the unique detected integration site. (F) In P10, 2 integrated copies at M+33 were detected, contrasting with the 1 copy detected before and after chemotherapy. These data confirmed that there is 1 leukemic clone bearing the 2 proviral integration sites, LMO2 and BMI1. (G–L) Number of integration sites shared (pink, yellow, and orange) versus not shared (gray) at the indicated time points for P7 (G–I) and P10 (J–L).