Evaluation of ISs in whole blood and integrated transgene abundance in bone marrow precursors. (A-B) IS analysis was done, using whole blood samples, by nonrestrictive linear amplification-mediated polymerase chain reaction [(nr)LAM PCR] and sequencing as described previously.²  (A) IS analysis for months 6 through 36 showing distribution of gene-marked cell clones. Each of the top 10 most represented unique ISs is indicated by a different color, with gray showing the cumulative proportion of all other unique ISs. The total number of ISs is indicated at the top of each column. None of the clones showed a clonal contribution of >30% of the total retrieved ISs. Month 36 (M36) visit 1 was the regular study follow-up. M36 visit 2 was at the time of MDS diagnosis. (B) IS clonal abundance for months 6 to 36. For individual samples, sequence data form all (nr)LAM-PCR amplicons were combined. RefSeq gene names for the top 10 genes located next to or at the IS are shown. None of the top 10 most prominent clones appeared in >2 samples collected at different time points. (C) A core BM biopsy was collected from the patient 20 days post-MDS diagnosis, fixed in formalin, decalcified, and routinely processed. (a) In situ hybridization (ISH) was performed on a tissue section using a specific probe to detect integrated transgene DNA, and immunohistochemistry (IHC) for CD235a (glycophorin A) protein was used to identify erythroid precursors. Red and black arrows indicate examples of the integrated transgene signal within erythroid (CD235a⁺) and nonerythroid (CD235a⁻) precursors, respectively. (b) A serial section stained with a probe specific to an absent gene (the bacterial gene Dapb) to control for nonspecific ISH signals and with the isotype control antibody to assess the background IHC signal. Counterstaining with hematoxylin was also performed. Scale bars, 20 μm.