Plasma cell enrichment from bone marrow samples was performed based on an algorithm described elsewhere (PMID 22706262). In all 90 MGUS cases and one MM sample with a low percentage of plasma cells in the initial sample, plasma cells were isolated from bone marrow by FACSAria (BD Biosciences, San Jose, CA, USA) system using CD138, CD19 and CD56 markers to obtain an abnormal plasma cell population with a purity >80%. In a total of 13 out of 90 MGUS patients, a normal plasma cell population with purity >95% was also included in the analysis. In the remaining 32 MM samples, plasma cells were obtained by anti-CD138 immunomagnetic beads (AutoMACS Pro, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) with a purity >75%.
Extracted molecule
genomic DNA
Extraction protocol
DNA was isolated using Gentra Puregene Kit or AllPrep DNA/RNA Micro Kit (both Qiagen, Hilden, Germany). MGUS DNA samples were amplified using the REPLI-g Midi/Mini Kit and purified by QIAamp DNA Mini Kit (both Qiagen) or by ethanol precipitation.
Label
Alexa Fluor 5
Label protocol
A total of 0.5-1.0/1.4-3.0 µg of unamplified/amplified tested and control DNA were fragmented by AluI/RsaI (Promega) enzymes and fluorescently labelled by BioPrime Total for Agilent aCGH Kit (Thermo Fisher Scientific) or treated by SureTag Complete DNA Labeling Kit (Agilent Technologies).
Plasma cell enrichment from bone marrow samples was performed based on an algorithm described elsewhere (PMID 22706262). In all 90 MGUS cases and one MM sample with a low percentage of plasma cells in the initial sample, plasma cells were isolated from bone marrow by FACSAria (BD Biosciences, San Jose, CA, USA) system using CD138, CD19 and CD56 markers to obtain an abnormal plasma cell population with a purity >80%. In a total of 13 out of 90 MGUS patients, a normal plasma cell population with purity >95% was also included in the analysis. In the remaining 32 MM samples, plasma cells were obtained by anti-CD138 immunomagnetic beads (AutoMACS Pro, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) with a purity >75%.
Extracted molecule
genomic DNA
Extraction protocol
As reference DNA, Human Genomic DNA (Promega, cat. G147A (male), cat. G152A (female), Madison, WI, USA) or Human Reference DNA (Agilent Technologies, included in cat. 5190-4240, Santa Clara, CA, USA) were used.
Label
Alexa Fluor 3
Label protocol
A total of 0.5-1.0/1.4-3.0 µg of unamplified/amplified tested and control DNA were fragmented by AluI/RsaI (Promega) enzymes and fluorescently labelled by BioPrime Total for Agilent aCGH Kit (Thermo Fisher Scientific) or treated by SureTag Complete DNA Labeling Kit (Agilent Technologies).
Hybridization protocol
Tumor and control DNA samples, together with COT Human DNA (Hoffman-La Roche, Basel, Switzerland) and hybridization mix (Oligo aCGH Hybridization Kit, Agilent Technologies) were co-hybridized to the arrays. All MGUS samples and 16 MM samples were tested by Agilent-029830 SurePrint G3 Human CGH+SNP 4x180K (Agilent Technologies). MM samples were tested either by Agilent-030587 CCMC CGH+SNP 180K (n=7), Agilent-021924 SurePrint G3 Human CGH 8x60K (n=5) or Agilent-031746 ISCA CGH 60K v2 (all Agilent Technologies) (n=5).
Scan protocol
DNA arrays were scanned using a Microarray Scanner (Agilent Technologies).
Description
Sample name: MM-020
Data processing
Feature Extraction Software v12.0.2.2 (Agilent Technologies) was used for data extraction and quality control evaluation. Genomic Workbench v7.0.4.0 (Agilent Technologies) was used for CNA calling and copy-number neutral loss of heterozygosity (cnnLOH) calling. CNAs were called by ADM-2 algorithm and following setting: ≥100 kbp size, ≥0.2 fold change of log2 ratio, ≥5 consecutive probes. CNAs were manually curated and the default Database of Genomic Variants (http://www.openhelix.com) for hg19 was used to eliminate common human population copy-number variants. CnnLOH areas larger than 5.0 Mbp were considered.
