Leukemia (2018) 32:2435–2444 https://doi.org/10.1038/s41375-018-0108-y ARTICLE Multiple myeloma, gammopathies Kinase domain activation through gene rearrangement in multiple myeloma 1 2 1 1 1 2 Gareth J Morgan ● Jie He ● Ruslana Tytarenko ● Purvi Patel ● Owen W Stephens ● Shan Zhong ● 1 1 1 1 1 2 2 Shayu Deshpande ● Michael Bauer ● Niels Weinhold ● Carolina Schinke ● Leo Rasche ● Mark Bailey ● Siraj Ali ● 2,3 2 2 1 1 1 Jeff Ross ● Vincent A Miller ● Phillip Stephens ● Sharmilan Thanendrarajan ● Maurizio Zangari ● Frits van Rhee ● 2,4 1 1 Tariq Mughal ● Faith E Davies ● Brian A Walker Received: 3 January 2018 / Revised: 8 February 2018 / Accepted: 2 March 2018 / Published online: 23 March 2018 © The Author(s) 2018. This article is published with open access Abstract Chromosomal rearrangements that result in oncogenic kinase activation are present in many solid and hematological malignancies, but none have been reported in multiple myeloma (MM). Here we analyzed 1421 samples from 958 myeloma patients using a targeted assay and detected fusion genes in 1.5% of patients. These fusion genes were in-frame and the majority of them contained kinase domains from either receptor tyrosine kinases (ALK, ROS1, NTRK3, and FGFR1)or cytoplasmic kinases (BRAF, MAP3K14, and MAPK14), which would result in the activation of MEK/ERK, NF-κB, or 1234567890();,: 1234567890();,: inflammatory signaling pathways. Fusion genes were present in smoldering MM, newly diagnosed MM, and relapse patient samples indicating they are not solely late events. Most fusion genes were subclonal in nature, but one EML4-ALK fusion was clonal indicating it is a driver of disease pathogenesis. Samples with fusions of receptor tyrosine kinases were not found in conjunction with clonal Ras/Raf mutations indicating a parallel mechanism of MEK/ERK pathway activation. Fusion genes involving MAP3K14 (NIK), which regulates the NF-κB pathway, were detected as were t(14;17) rearrangements involving NIK in 2% of MM samples. Activation of kinases in myeloma through rearrangements presents an opportunity to use treatments existing in other cancers. Introduction targeted by specific tyrosine kinase inhibitors (TKIs), resulting in clinically relevant responses [3, 4]. Fusion genes are the product of genomic rearrangements In solid tumors, fusion genes are also seen and similarly where two genes are rearranged to create a new fusion gene can result in the activation of kinase domains with key with either increased or inappropriate expression and deregulated genes including the receptor tyrosine kinases functionality that was not previously evident. One of the (RTKs) ALK, ROS1, RET, FGFR1/2/3, and NTRK1/2/3,as initial rearrangements defining fusion genes was the t(9;22), well as cytoplasmic kinases such as BRAF [5, 6]. These typical of chronic myeloid leukemia (CML) cases [1, 2]. rearrangements are not as frequent as the t(9;22) with rates This translocation generates a BCR-ABL1 fusion gene, of up to 12% in patients with thyroid carcinoma, but on the which results in the activation of the tyrosine kinase domain whole, across all types of cancer, the rate is in the order of of the ABL protein. The increased signaling, which occurs 1–2% [7]. The most common signaling pathway deregu- as a result of this rearrangement, can be therapeutically lated by these fusion genes is the MEK/ERK pathway. Analogous to CML, these kinase fusion genes can also be therapeutically targeted, a key example of which is the * Brian A Walker EML4-ALK fusion gene seen in 3–7% of patients with [email protected] adenocarcinoma of the lung [8]. Multiple myeloma (MM) is characterized by primary 1 The Myeloma Institute, University of Arkansas for Medical translocations into the immunoglobulin (Ig) loci, which occur Sciences, Little Rock, AR, USA in ~40% of patients [9]. These structural rearrangements place 2 Foundation Medicine Inc., Cambridge, MA, USA the Ig super-enhancer next to an oncogene, resulting in its 3 Albany Medical College, Albany, NY, USA overexpression [10].ThecommongenefusionsseeninMM 4 Tufts University Medical Center, Boston, MA, USA involve the Ig loci, including cases with a t(4;14) where the 2436 G. J. Morgan et al. breakpoint within MMSET results in an Ig-MMSET transcript, rearrangements. Variant processing is described elsewhere but an alteration in functionality as a consequence is not seen [14, 16], but importantly involved removal of germ line [11, 12]. A key signaling abnormality in MM is increased variants from the 1000 Genomes Project (dbSNP135), as a MEK/ERK pathway activation due to activating point muta- matched patient non-tumor sample is not used to identify tions in KRAS, NRAS,andBRAF seen in up to 50% of patients truly somatic variants. All inactivating events (i.e., trunca- [13]. However, despite the frequent deregulation of this path- tions and deletions) in known tumor suppressor genes were way, functional gene fusions involving RTKs have not been also called as significant. To maximize mutation-detection described. Here we analyzed 1421 patient samples using a accuracy (sensitivity and specificity) in clinical specimens, targeted assay able to detect gene fusions and describe their the test has been optimized and validated to detect base prevalence in MM. substitutions at a ≥5% variant allele frequency (VAF) and indels with a ≥10% VAF to ≥99% accuracy. However, mutations are reported down to 1% VAF where the variant Methods is a known hotspot and there is sufficient purity and sequencing depth. Reports were generated by Foundation Patient samples and nucleic acid extraction Medicine and data files containing additional information (VAF, variant type, depth at variant location, genomic We report on 1421 samples from 958 individuals diagnosed coordinates) were received. with monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), or MM who RT-PCR confirmation of fusion genes underwent targeted sequencing with the FoundationOne heme (F1H) assay [14, 15] between September 2013 and August To confirm the fusion gene breakpoints, reverse 2016. All patients signed a written informed consent in transcription-polymerase chain reaction (RT-PCR) was keeping with institutional, federal, and Helsinki Declaration performed on seven samples. Complementary DNA guidelines. Tumor samples were obtained from bone marrow (cDNA) was synthesized from 200 ng RNA using iScript aspirates, enriched by CD138+ selection using magnetic (Bio-Rad). Two microliters of a 1/10 dilution of the cDNA beads (AutoMACs, Miltenyi Biotech, Cologne, Germany or was used in subsequent PCR reactions. PCR bands were RoboSep, StemCell Technologies, Vancouver, Canada). excised from agarose gels, purified, and sequenced by the RNA and DNA were extracted using the AllPrep DNA/RNA Sanger method. Sanger sequence reads were compared mini kit (Qiagen, Hilden, Germany), RNeasy RNA extraction against the genome using BLAST. kit (Qiagen), or Puregene DNA extraction kit (Qiagen). Determining clonality of fusion genes Foundation One heme reporting Custom probe-based qPCR assays (Integrated DNA Tech- DNA ≥50 ng and/or RNA >300 ng was interrogated using nologies) were designed across the DNA breakpoints in the F1H Panel (Foundation Medicine, MA). The panel order to determine if the fusion genes are clonal. Assays analyzes the complete coding DNA sequence of 405 genes, were also designed against the unarranged germ line as well as selected introns of 31 genes involved in chro- sequence for comparison, as well as for any Ig transloca- mosomal rearrangements. It also interrogates the RNA tions that had also been detected in the same samples. Only sequence of 265 commonly rearranged genes resulting in samples with an identified DNA breakpoint were analyzed gene fusions. Genes included in this assay encode known or as the sequence is required to design the assays. Rearranged likely targets of therapy, either FDA-approved or in clinical and germ line sequence assays were labeled with FAM or trials, or are otherwise known drivers of oncogenesis. Due HEX, respectively. DNA from the samples was used in a to the capture strategies, most fusions are detected by either digital droplet PCR reaction to determine the proportion of DNA or RNA sequencing and not both. Of the 1421 sam- sample with a rearrangement. If a primary IGH transloca- ples, 565 were processed on the DNA panel only and 856 tion breakpoint was assayed, it was assumed to be clonal were processed on the DNA and RNA panels. Data have and the percent positive droplets in the IGH and fusion gene been submitted to the European Genome-Phenome Archive assay adjusted to account for CD138 cell purity. under accession EGAS00001002874. Sequencing was carried out to an average depth of 468x Gene expression profiling and was performed using a HiSeq 2500 (Illumina). Sequences were analyzed for base substitutions, indels, Gene expression profiling (GEP) using Affymetrix U133 copy-number alterations (focal amplifications with ≥8 Plus 2.0 arrays was performed. The GEP-based 70 risk copies and homozygous deletions), and selected gene score (GEP70) and molecular subgroups were determined Kinase domain activation through gene rearrangement in multiple myeloma 2437 Table 1 In-frame fusion genes identified by RNA or DNA sequencing Sample Disease state Cytogenetic group GEP70 risk Head gene Head last Tail gene Tail first In-frame RNA-seq RT-PCR DNA-seq ddPCR Fusion gene group exon exon function 35882 SMM t(4;14) LR TSPAN3 5 ROS1 31 Yes Yes Yes NC ND Kinase 38203 NDMM HRD LR ATM 57 DLG2