Rapid, Robust and Accessible Molecular Profiling of Biologically and Clinically Relevant Copy Number Alterations in Multiple Myeloma from Small Amounts of Tumor DNA
Authors: Kaiser Martin F , Proszek Paula , Begum Dil , Dahir Nasrin , Walker Brian A , Savola Suvi , Boyle Eileen M , Johnson David C , Cairns David A , Gregory Walter , Jackson Graham , Owen Roger G , Cook Gordon , Drayson Mark T , Davies Faith E , Morgan Gareth J 
Published: October 2019

Abstract
Background
Structural aberrations like copy number alterations (CNA) and translocations play a central role in multiple myeloma tumor biology. The analysis of CNA for complete molecular profiling of myeloma retains clinical significance in the context of recent next-generation sequencing (NGS) results which highlight the highly heterogeneous landscape of single nucleotide mutations, which are often sub-clonal. Detection of CNAs remains the domain of array-based technologies, as demonstrated by the use of copy number arrays by the TCGA consortium and others for CNA detection in NGS projects. High cost and infrastructure attached to CNA analysis by array limit access to this technology in many molecular diagnostic laboratories. We present here a robust and accessible method that combines two multiplex ligation-dependent probe amplification (MLPA) assays for the relevant CNAs in myeloma using low input amounts of purified tumor DNA.
Methods
Bone marrow myeloma cells from patients at presentation and at relapse from the Myeloma IX and Myeloma XI trials were purified to >95% purity by immunomagnetic separation (Miltenyi Biotech) and DNA was extracted using AllPrep columns (QIAGEN) for analysis by MLPA. A novel myeloma specific probemix was designed and technically validated by MRC-Holland (Amsterdam, The Netherlands), complementing the established myeloma specific probemix P425 (Alpar et al., Gen Chrom Cancer 2013).
Results
In total 130 purified myeloma cell DNA samples were successfully analyzed by MLPA, interrogating 43 different loci in the following regions: 6p22 - 6p12 [incl. CDKN1A]; 6q12 - 6q26 [incl. TNFAIP3, WTAP, PARK2]; 8p23 – 8p11 [incl. TNFRSF10A/B]; 8q12 – 8q24 [incl. MYC]; 11q13 – 11q25 [incl. CCND1, BIRC2, BIRC3]; 17p [all exons of TP53]; 22q11 – 22q13 [incl. HIRA, EP300]. The assay contains one probe for the detection of BRAF V600E, including determination of mutant allele ratio. This probemix complements another MLPA assay [P425] which interrogates prognostically and biologically relevant regions such as 1p32, 1q21-1q23, 13q14, 16q12-16q23, 17p13 and chr 5, 9 and 15 for detection of hyperdiploidy (HRD). The assay returned good quality results using as little as 25 ng DNA input material without modification of assay conditions.
In this group of cases which were enriched with cases known to carry specific CNAs and mutations, frequent copy number aberrations encompassed loss of chr(6q), in the majority of cases (16%) affecting all genes investigated in the region 6q23 – 6q26. In addition, five cases (4%) with an isolated heterozygous deletion of TNFAIP3 at 6q23 and two cases with deletion of TNFAIP3 and WTAP were detected. Gains of MYC at 8q24 were observed in 13 cases (10%) of which 5 showed gains of other regions on 8q as well. BIRC3 and BIRC2 were both lost in 4 cases, including 1 homozygous deletion of both genes. All coding exons of TP53 were heterozygously deleted in 16% of cases in this group containing specifically selected relapsed cases. Gain of chromosome 11 was frequent (29%) and mostly associated with HRD, but gain(11q) without HRD was observed, including 3 cases with isolated gain of the CCND1locus at 11q13.3.
Of 11 cases with a known BRAF V600E mutation identified by SSCA, 10 (91%) were detected by MLPA with the remaining mutation showing a signal just above detection limit by SSCA. Calculated median V600E mutant allele ratio in comparison to an artificial plasmid mix mimicking a heterozygous mutation was 39% (range 9%-77%), confirming our previous observation that this mutation is mostly heterozygous and sub-clonal in myeloma.
Further cases at presentation from the Myeloma XI trial are currently being analyzed by MLPA and results will be presented at the meeting, including a comparison with matched CNA array data for a selection of cases as well as correlation with clinical data for trial cases.
Discussion
The combination of this panel with another MLPA probemix [P425], which has been extensively validated in over 1200 myeloma cases by our group, allows assaying of biologically and clinically relevant CNAs in myeloma as well as BRAF V600E mutation status. The tests can be run on standard laboratory equipment and require 25 ng of DNA as input material, making this method suitable for exploring the clinical impact of myeloma specific CNAs in large clinical trials with a potential of transferring the method to the routine diagnostic setting.

Disclosures
Walker: Onyx: Consultancy. Savola:MRC-Holland: Employment.