RNA-seq is a valuable complement of conventional diagnostic tools in newly diagnosed AML patients
Authors: Rocco Piazza, Caterina Cecchetti, Alessandra Pirola, Carla Donandoni, Diletta Fontana, Caterina Mezzatesta, Vera Magistroni, Benedetta Bianchi, Lorenza Borin, Monica Fumagalli, Carlo Gambacorti‐Passerini
Journal: American Journal of Hematology (2015)

Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults. The identification of specific genetic alterations in AML is useful for both prognosis and treatment choice. In recent years, thanks to the development of “Next-Generation Sequencing” technologies (NGS), the list of molecular markers able to impact on prognosis and treatment of AML grew very quickly. Moreover, the number of clinically available targeted inhibitors increased at a very fast pace. Despite the availability of these new tools, cytogenetic analysis remains the standard de facto to detect chromosomal abnormalities. However, this technique suffers from several limitations, such as the requirement of an adequate number of mitotic cells, the limited resolution, and the inability to detect cryptic fusions. Similarly, genotyping performed with standard techniques, such as PCR/Sanger sequencing, is time consuming, expensive and allows to detect only a predefined set of known variants. The result is that only a limited fraction of the known oncogenic lesions is routinely tested in clinical laboratories. In the present study, 20 AML patients at diagnosis were subjected to RNA-Seq to identify gene fusions, NPM1 insertions, FLT3 internal tandem duplications, and single nucleotide variants (SNV) and the results were compared with those obtained by the routine clinical approach. Samples were processed as described in the Supporting Information Methods. The libraries were sequenced on an Illumina HiSeq 2500 or a Genome Analyzer IIx with 76bp paired-end reads. FastQ sequences were aligned to the human genome (GRCh38/hg38) using Star 1 and processed with Samtools 2. Bam files were analyzed using CEQer2, an evolution and integration of FusionAnalyser 3 and CEQer 4. Given that RNA-Seq analysis was performed only on leukemic samples, variant analyses were restricted to a predefined list of genes involved in AML pathogenesis (CEBPA, NPM1, FLT3, RUNX1, KMT2A, WT1, EZH2, NF1, EVI1, KIT, H-RAS, K-RAS, TET2, IDH1, IDH2, DNMT3A, BCOR, BCORL1, NUP98, ASXL1, ABCB5, BAALC, CEP72, DIP2C, ROBO1, KLC1, TP53, IGFBP7, SETBP1, JAK2, NRAS, NOTCH1, CDKN2A, MPL, SF3B1, BRAF, PTPN11, SRSF2, IKZF1, GATA1, MYD88, ATM, CBL, PHF6, BCL2) to minimize the risk of identifying rare SNPs. In 20 patients, a total of nine fusions were identified (Table 1). Of them, five were previously detected by cytogenetic analyses (one RUNX1-RUNX1T1 [AML-ETO], three PML-RARA, and one CBFB-MYH11). Of the four fusions detected only by RNA-Seq, one was completely new (ETV6-KDM2B) and three were reported in previous works (ETS2-ERG, KMT2A-MLLT10, and ZMYM2-FGFR1). ETV6-KDM2B, resulting from t(12;12)(p13;q24), is a bona fide intrachromosomal inversion involving the telomeric regions of the long (KDM2B) and short (ETV6) arm of chromosome 12 and defining a previously unreported in-frame cryptic translocation (Supporting Information Fig. 1). ETS2-ERG is a cryptic, in-frame intrachromosomal translocation occurring in chromosome 21 3. KMT2A-MLLT10, also known as MLL-MLLT10, is an in-frame recurrent translocation resulting from t(10;11)(p12;q23) and characterized by poor prognosis 5. ZMYM2-FGFR1 is particularly interesting, because it defines the 8p11 myeloproliferative syndrome, known also as Stem Cell Leukemia/Lymphoma, an extremely rare stem cell disorder characterized by very poor prognosis and association of myeloproliferative neoplasm with T/B-cell lymphoma. Notably, this fusion causes constitutive activation of the FGFR1 kinase and, therefore, it is potentially targetable using drugs such as ponatinib 6. Indeed, in vitro treatment of patient's cells with nanomolar concentration of ponatinib caused a potent and specific growth inhibition (Supporting Information Fig. 2). A total of three NPM1 insertions were reported by both standard techniques and RNA-Seq; FLT3-ITD was detected in six samples by RNA-Seq and in four by standard PCR-based techniques (not tested in the remaining two). Although the numbers are too limited to drive definitive conclusions, this suggests comparable sensitivity of standard techniques and RNA-Seq in detecting these variants (Table 1). A total of 23 SNV were also identified by RNA-Seq with a median of one variant per patient (range 0–4). Globally, 22/23 SNV have been confirmed by Sanger sequencing, suggesting that the accuracy of our pipeline for SNV detection is 96%. Of the 22 confirmed SNV, 10 were new and 12 were known variants (Table 1). Taken globally, these data suggest that a single RNA-Seq analysis performed at diagnosis in AML patients, even in absence of a matched control sample, allows to quickly detect a large number of abnormalities that are not detected using standard clinical tests. This analysis will allow to achieve a better prognostic assessment and to identify potentially new treatment options, such as ponatinib for the patient carrying the ZMYM2-FGFR1 translocation. This is of paramount importance, as the list of clinically available selective inhibitors keeps growing at very fast pace and the discovery of new driver variants may lead to the development of novel targeted therapies (Supporting Information Table I). Rocco Piazza1*, Caterina Cecchetti2*, Alessandra Pirola1, Carla Donandoni1, Diletta Fontana1, Caterina Mezzatesta1, Vera Magistroni1, Benedetta Bianchi1, Lorenza Borin2, Monica Fumagalli2, and Carlo Gambacorti-Passerini1,2 1Department of Medicine and Surgery, University of Milano-Bicocca, Monza, Italy and 2 Hematology, San Gerardo Hospital, Monza, Italy Additional Supporting Information may be found in the online version of this article. Supporting Information Figure 1. Supporting Information Figure 2. Supporting Information Table 1. Supporting Information Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.