Morphologic features of the APL case and characterization of the TTMV/RARA chimeric fusion transcript. (A) Morphology of the case of APL, negative for RARA rearrangement. Nuclear size and shape are irregular, whereas the cytoplasm is for the most part occupied by densely packed granules. (B) Average relative expression level from WTS data of RARA exons 2 and 3, and the expressed intronic region is shown for the index case (blue squares) and for another 12 AMLs with known chromosomal aberrations. (C) Schematic representation of the fusion between the TTMV sequence and RARA exon 3. The chimeric transcript shows the conserved TTMV UTR sequence followed by the ORF2, immediately upstream of the short sequence of retained RARA intron 2, in frame with the full RARA exon 3. A new splicing event that joins this part of the retained intron 2 to exon 3 and removes the intervening intronic sequence occurs during splicing. Some representative sequencing reads that support the presence of the fusion transcript are shown. The exon–intron gene structure and the location of the integrated viral sequence is indicated at the bottom. (D-E) Sanger sequencing electropherograms of the downstream (D) and upstream (E) breakpoints of TTMV integration on the leukemia relapse sample genomic DNA. (F) Detection of viral integration with primers common to the 3 Anelloviruses TTV, TTMDV, and TTMV (NG779/NG782), and those specific only for TTMV (NG792/NG791), in genomic DNA of the relapsed APL case and in another 6 AML samples. (G-H) Detection of the TTMV/RARA chimeric fusion in genomic DNA (G) and cDNA (H) in sequential samples of the APL case from diagnosis to relapse, during remission–induction therapy, and after HSCT. (I) Quantitative real-time PCR detection of the TTMV/RARA fusion transcript decrease during induction therapy and after HSCT.