Genomic analysis of therapy‐related acute promyelocytic leukemias arising after malignant and non‐malignant disorders
Authors: Tiziana Ottone, Syed Khizer Hasan, Maria Teresa Voso, Antonio Ledda, Enrico Montefusco, Susanna Fenu, Maria Pagoni, Maximillian Hubmann, Monia Lunghi, Uwe Platzbecker, Francesco Lo‐Coco
Journal: American Journal of Hematology (2013)

Therapy-related acute promyelocytic leukemia (t-APL) has similar clinico-biological characteristics and treatment outcome as compared to de novo APL 1. In the past 2 decades, several in vitro and in vivo studies have elucidated the molecular mechanisms underlying APL arising as de novo while only few studies have investigated the pathophysiology of t-APL 2. Chemotherapeutic drugs given for primary disorders usually include topoisomerase II inhibitors which can increase the risk of development of therapy-related leukemia 3. Among newly diagnosed APLs, the frequency of t-APL is 10–12% approximately and most of the reported t-APL patients received topoisomerase II inhibitors for the treatment of primary disease 2, 4. Genomic sequence analysis at breakpoint junction of PML/RARA in t-APL may help to elucidate the mechanism of t(15;17) by relating specific genetic abnormalities to the biologic effects of cytotoxic agents given for primary disease. Previously, we showed that specific genomic loci within the genome, for example PML intron 6 and RUNX1 intron 5, contain preferential sites of topoisomerase II mediated DNA cleavage in the presence of its inhibitor 5, 6. Here we report genomic breakpoint characterization of 12 unpublished cases of t-APL in which non-malignant (n = 7) and malignant (n = 7) disorders accounted for the primary disease. DNA samples at the time of t-APL diagnosis were collected through collaboration with Hematology institutes located in Germany, Greece, and Italy. To identify the precise location of breakpoints at PML and RARA loci, we adopted a long-range PCR strategy followed by direct DNA sequencing as reported elsewhere 4. Patient's main demographic and clinical characteristics, primary disease, precise location of breakpoints at PML, RARA loci, and PML/RARA isoforms are reported in Table 1. Seven of 12 patients received topoisomerase II targeting agents for the treatment of primary disease. By comparing RARA intron 2 breakpoint data of the present series with the previously reported cases of t-APL 7, we observed five RARA intron 2 breakpoints within 1–3 bp of one another (pts 2, 4, 5, 7, and 10). Patient 7 was diagnosed with Lewis–Sumner syndrome, a multifocal demyelinating neuropathy, did not receive topoisomerase II inhibitors before APL development. The common RARA breakpoint at nucleotide position 14919–25 in pt 7 with previously reported case of multiple sclerosis who received only interferon-β suggests the presence of a natural topoisomerase II cleavage site in this region of DNA. Chromosome 15 breakpoints in PML intron 6 were located in three cases (pts 3, 5, and 6) in a previously identified “hotspot” region (Table 1). Three out of 5 bcr1 positive patients who received mitoxantrone displayed clustering of breakpoints within an 8 base bp “hotspot” region. We first time identified a patient (pt 5) in whom both PML and RARA breakpoints were located within the two previously identified breakpoint “hotspot” regions. These “hotspots” (PML 1482–89 and RARA 14446–49) have been shown in functional assays as preferential sites of DNA cleavage mediated by topoisomerase II 4, 5. In conclusion, genomic characterization of 12 previously unreported cases of t-APL confirms the presence of hotspots at the DNA level on both RARA and PML sites. In addition, our data may serve as a reference for future genomic studies on t-APL to identify preferential breakpoints at these loci in the presence or absence of topoisomerase II targeting agents given for primary malignant or non-malignant disease. TO and SKH carried out the experiments, analyzed the data and wrote the manuscript; MTV, AL, MP, MH, ML, UP, SF and EM provided patient's samples; FLC designed the study, supervised the research and wrote the manuscript. All authors read and approved the final version of the manuscript. Tiziana Ottone,1,2 Syed Khizer Hasan,1,2 Maria Teresa Voso,3 Antonio Ledda,4 Enrico Montefusco,5 Susanna Fenu,6 Maria Pagoni,7 Maximillian Hubmann,8 Monia Lunghi,9 Uwe Platzbecker,10 Francesco Lo-Coco1,2* 1Department of Biopathology, University of Tor Vergata, Rome, Italy 2Laboratorio di Neuro-Oncoematologia, Fondazione Santa Lucia, Rome, Italy 3Istituto di Ematologia, Università Cattolica del Sacro Cuore, Rome, Italy 4Ematologia/Centro Trapianti Midollo Osseo, Ospedale R. Binaghi, Cagliari, Italy 5Faculty of Medicine and Psychology, Sapienza University, Rome, Italy 6Department of Hematology, Azienda Ospedaliera San Giovanni-Addolorata, Roma, Italy 7Department of Hematology and Lymphoma, Evangelismos Hospital, Athens, Greece 8Ludwig-Maximillians University Munich, Grosshadern Internal Medicine, Munich, Germany 9Department of Clinical and Experimental Medicine, Università del Piemonte, Orientale Amedeo Avogadro, Novara, Italy 10Universitätsklinikum Department of Medicine, Carl Gustav Carus, Dresden, Germany