Balkan Journal of Medical Genetics

PROS AND CONS FOR FLUORESCENT IN SITU HYBRIDIZATION, KARYOTYPING AND NEXT GENERATION SEQUENCING FOR DIAGNOSIS AND FOLLOW-UP OF MULTIPLE MYELOMA
Ikbal Atli E, Gurkan H, Onur Kirkizlar H, Atli E, Demir S, Yalcintepe S, Kalkan R, Demir AM
*Corresponding Author: Assistant Professor Emine Ikbal Atli, Department of Medical Genetics, Faculty of Medicine, Trakya University, Balkan Campus, Highway D100, Edirne, Turkey 22030. Tel: +284-235- 76-41/23-30. Fax: +284-235-86-52. E-mail: eikbalatli@trakya.edu.tr
page: 59

MATERIALS AND METHODS

The present study includes 35 patients (17 males and 18 females) from April 2018 to December 2019. Written informed consent was obtained from all subjects after a full explanation of the study protocol, which was approved by the Ethics Committee of the Faculty of Medicine, Trakya University, Erdine, Turkey, and conducted in accordance with the ethics principles established by the Declaration of Helsinki. Cytogenetic Evaluation. Cytogenetics studies were carried out either on bone marrow or peripheral blood of patients. Diagnoses were based on the International Myeloma Working Group (IMWG) definition of MM [5]. The morphology of patient bone marrow specimens confirmed a myeloma in all cases. Chromosomal analysis was performed on cultured bone marrow samples using a standard G-banding technique. At least 20 metaphase spreads were analyzed and results were reported according to the International System for Human Cytogenetic Nomenclature (2016) [9]. Fluorescent In Situ Hybridization Analysis. In order to analyze molecular cytogenetic abnormalities, inter phase FISH was applied to non dividing cells, according to the manufacturer’s instructions (Cytocell, Cambridge, Cambridgeshire, UK). The list of analyzed loci is as follows: IGH/FGFR3 Dual Fusion (DF) Probe (Cytocell) for (4;14) (p16.3; q32.33); IGH/MAF DF Probe (Cytocell) for t(14;16) (q32.33; q23.1); IGH/CCND1/MYEOV DF Probe (Cytocell) for t(11;14)(q13;q32.33); 13q14.3 Dual Color Probe (Cytocell) for del(13q14); P53 (TP53) Dual Color Probe (Cytocell) for del(17p13); ATM Dual Color Probe (Cytocell) for del(11q22.3). The evaluation of FISH signals was performed using a fluorescence microscope (Axio Imager, M1; Carl Zeiss, Göttingen, Germany) with the software Cytovision 3.6 (Leica Biosystems Inc., Buffalo Grove, IL, USA). At least 200 inter phase nuclei were analyzed for each slide. Next Generation Sequencing. Next generation sequencing technology allows millions of sequence readings to be processed in parallel; this makes it an excellent tool for identification of low-rate clonality detection. In fact, this technology is used not only to cover large genomic regions, but also for ultra-deep sequencing of small genomic regions, so it can be used for assessment of clonal rearrangements of MM-related markers such as the immunoglobulin heavy chain locus (IgH) gene [10]. DNA was isolated from bone marrow (QIAamp DNA Blood Mini Kit; Qiagen GmbH, Hilden, Germany) and quantified using a Qubit fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). Multiple myeloma specific target genes were selected as shown in Table 1. Myeloid Panel QIAseq Targeted DNA Panel (Qiagen GmbH) was used to be able to enrich genes and construct libraries for NGS analysis for 141 genes that are commonly mutated in MM. This panel covers exonic regions of genes and ±5-10 bases of exon/intron boundaries. The panel was designed to cover genes based on library fragments of 250 bp produced from high-quality DNA samples (Table 1). Amplified targets were sequenced on the NextSeq (Illumina Inc., San Diego, CA, USA) sequencer according to the manufacturer’s protocol for paired-end sequencing. The ingenuity variant analysis (IVA) program was used for data analysis and quality assessment of SNVs, short insertions and deletions. Multiple databases were consulted when performing variant analysis of patients [American College of Medical Genetics and Genomics (ACMG) (www.acmg.net), Bethesda, MD, USA [11]; COSMIC (https://cancer.sanger.ac.uk/cosmic); HGMD (www.hgmd.cf.ac.uk.ac.index.php); ClinVar (www.ncbi. nlm.nih.gov.clinvar/); Uniprot (www.uniprot.org/); Genom AD (https//gnomad.broadinstitute.org/); Varsome (https:// varsome.com); Franklin (https://flanklin.genoox. com/clinical- db/home)]. Amplicons were considered as dropout and excluded from analysis if the coverage at any analyzed position in any of the two-paired end sequences (minimal coverage) was <80 reads. Validity of somatic mutations was checked against publicly accessible databases. Variants detected at a coverage of >100 ×, with allele frequency >5.0% were included for subsequent investigation. Known hotspots or clinically actionable variants detected below these thresholds were verified using orthogonal methods such as Sanger sequencing.

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