Balkan Journal of Medical Genetics

CREBBP IS A MAJOR PROGNOSTIC BIOMARKER FOR RELAPSE IN CHILDHOOD B-CELL ACUTE LYMPHOBLASTIC LEUKEMIA: A NATIONAL STUDY OF UNSELECTED COHORT
Krstevska Bozhinovikj E1, Matevska-Geshkovska N1, Staninova Stojovska M1, Gjorgievska E1, Jovanovska A2, Kocheva S2*, Dimovski A1,3*
*Corresponding Author: *Corresponding Authors: Prof. Aleksandar Dimovski MD PhD. Center for Biomolecular Pharmaceutical Analyses, Faculty of Pharmacy, University Ss. Cyril and Methodius in Skopje, Mother Theresa 47, 1000 Skopje, N. Macedonia adimovski@ff.ukim.edu.mk; phone number: +38923119694 ext109; Research Center for Genetic Engineering and Biotechnology “Georgi D. Efremov”, Macedonian Academy of Sciences and Arts, Bul. Krste Misirkov 2, 1000, Skopje, N. Macedonia, a.dimovski@manu.edu.mk; phone number: +38923235411
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MATERIALS AND METHODS

Clonal rearrangements detection and MRD analysis All patients (a total of 55) included in the study were diagnosed with B-ALL at the University Clinic for Pediatric diseases in Skopje over a period of 6 years (2018-2023). For the molecular clonality analyses, nucleic acids were extracted from bone marrow mononuclear cells using an au- tomated nucleic acid extractor (MagCore Super, RBC Bio- science, New Taipei City). The clonal immunoglobulin (Ig) gene rearrangements were identified using multiplex PCR according to the BIOMED-2 protocol [16]. The dominant rearrangements (from the diagnostic and relapse samples) were subsequently sequenced with the specific family prim- ers using the Sanger sequencing method, and the sequences were analyzed using the IMGT/V-QUEST and IgBLAST tools. The initial clonal rearrangements were also detected using Next-Generation Sequencing (NGS) method with the LymphoTrack Dx IGH-FR3 assay (Invivoscribe Technolo- gies, San Diego, CA, USA) according to the manufacturer’s protocol. These clones were tracked for MRD analysis at two time-points, on day 33 and day 78 from therapy onset, with a sensitivity threshold at 10^(-4) [17]. Hybrid transcripts and copy number alterations The most common gene rearrangements, includ- ing t(12;21)(p13;q22) ETV6::RUNX1, t(1;19)(q23;p13) TCF3::PBX1, t(9;22)(q34;q11) BCR::ABL1, and t(4;11) (q21;q23) MLL::AFF1 were detected using qRT-PCR according to the BIOMED-1 protocol [18]. Aneuploidy was assessed using the SALSA MLPA P036 and P070 subtelomeric probe-mix kits (MRC-Holland, Amsterdam, the Netherlands) following the manufacturer’s recom- mended procedures. High-hyperdiploidy or hypodiploidy were defined if >50 or <44 chromosomes were present, respectively. Identification of copy number alterations in specific regions and genes with prognostic significance in B-ALL was performed using the P327, P329, P335, and P038 SALSA MLPA kits, which allowed for the detection of intrachromosomal amplification of chromosome 21 (iAMP21), EGR deletion, deletions or duplications in the PAR1 region (CRLF2, CSF2RA, IL3RA), deletion of TP53, deletion of IKZF1, and deletions or duplications in genes associated with B-cell differentiation and cell cycle control (CDKN2A/2B, PAX5, ETV6, RB1, BTG1, and EBF1). Whole exome sequencing Targeted, massively parallel sequencing of exons in >99% of protein-coding genes (Whole Exome Sequencing, WES) was performed in all patients with relapse (samples obtained both at diagnosis and at relapse) and in five ad- ditional patients in remission. The reactions were con- ducted on an Illumina NovaSeq 6000 sequencer, using the Twist Human Core + RefSeq + Mitochondrial Panel kit (Twist Bioscience, San Francisco, CA, USA) with a mean read depth of targeted regions across samples of approximately 100X. Variant annotation, filtering, and classification of the detected variants were done accord- ing to ACMG guidelines, utilizing multiple databases and tools (ClinVar, HGMD, dbSNP, ExAC, gnomAD, OMIM, Varsome, Franklin Genoox). The patients were treated according to the ALL-IC- BFM 2002 protocol, and the median follow-up was 46 months (range: 11-79). Since this was an observational study, the MLPA analyses and the NGS-MRD data were not used for patient stratification or clinical decisions. The study was approved by the Ethics Subcommittee of the Macedonian Academy of Sciences and Arts, and written informed consent was obtained from the patients’ guard- ians in accordance with the Declaration of Helsinki.

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