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1605 Distinct Pattern of ABL1 Genomic Breakpoints in Chronic Myeloid Leukemia and BCR::ABL1-Positive Acute Lymphoblastic Leukemia: Analysis of 884 Patients with Minor and Major BCR::ABL1 Fusion

Program: Oral and Poster Abstracts
Session: 618. Acute Lymphoblastic Leukemias: Biomarkers, Molecular Markers and Minimal Residual Disease in Diagnosis and Prognosis: Poster I
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Lymphoid Leukemias, ALL, adult, Translational Research, elderly, CML, genomics, Chronic Myeloid Malignancies, hematopoiesis, pediatric, Diseases, Lymphoid Malignancies, young adult , Myeloid Malignancies, Biological Processes, molecular biology, Study Population, Human
Saturday, December 9, 2023, 5:30 PM-7:30 PM

Lenka Hovorkova, MSc, PhD1*, Lucie Winkowska, PhD1*, Justina Krotka, MD1*, Manuela Krumbholz2*, Claus Meyer, PhD3*, Rosemary Sutton, BSC, PhD4*, Michelle Henderson, BSc(hons), PhD4*, Emmanuelle Clappier, PhD, PharmD5*, Sabina Chiaretti, MD, PhD6, Lucie Sramkova, MD, PhD7*, Jan Stary, MD, DSc7*, Adela Benesova, PhD8*, Katerina Machova Polakova8*, Rolf Marschalek, PhD3*, Markus Metzler, MD9*, Giovanni Cazzaniga, PhD10, Gunnar Cario, MD11*, Jan Trka, MD, PhD12, Marketa Zaliova, MD, PhD13* and Jan Zuna, MD, PhD13

1CLIP, Department of Pediatric Hematology and Oncology, Second Faculty of Medicine, Charles University/University Hospital Motol, Prague, Czech Republic
2Pediatric Oncology and Hematology, Department of Pediatrics and Adolescent Medicine, University Hospital, Erlangen, Germany
3Institute of Pharmaceutical Biology/Diagnostic Center of Acute Leukemia, Goethe-University, Frankfurt, Germany
4Children's Cancer Institute, Randwick, Australia
5Hôpital Saint-Louis, APHP, Paris, FRA
6Division of Hematology, Department of Translational and Precision Medicine, Division of Hematology, Rome, Italy
7Department of Pediatric Hematology and Oncology, Second Faculty of Medicine, Charles University/University Hospital Motol, Prague, Czech Republic
8Institute of Hematology and Blood Transfusion, Prague, Czech Republic
9Department of Pediatrics and Adolescent Medicine, University Hospital Erlangen, Erlangen, Germany
10Clinica Pediatrica Univ. Milano Bicocca Ospedale San Gerardo, Monza, MB, ITA
11Department of Pediatrics, University Hospital Schleswig-Holstein, Kiel, Germany
12Charles University, Prague, Prague, Czech Republic
13CLIP, Department of Pediatric Hematology and Oncology, University Hospital Motol, Prague, Czech Republic

The BCR::ABL1 fusion gene is a hallmark of chronic myeloid leukemia (CML) but is also found in patients with acute lymphoblastic leukemia (ALL). There are two main breakpoint clusters in BCR – “minor” (between exons 1-2, resulting in p190 fusion protein, prevalent in ALL and scarce in CML) and “Major” (between exons 13-15, resulting in p210 protein). On the ABL1 side, the breakpoints are mostly localized between exons 1-3. Due to large intronic areas where the breakpoints occur, only a few papers have been published focusing on the structure of BCR::ABL1 fusions. Moreover, with a single exception, these studies included only the Major-BCR::ABL1 patients. Here, we focused on the analysis of BCR::ABL1 breakpoints involving CML and ALL, children and adults, and Major and minor forms of fusion. To our knowledge, we present data on the largest cohort of patients with BCR::ABL1 fusion identified at the DNA level described to date.

Genomic breakpoints were found in 884 patients with BCR::ABL1+ ALL (n=463) and CML (n=421) by multiplex long distance PCR/Sanger sequencing or by NGS Custom Target Enrichment. The RSS database, MEME software and RepeatMasker were used to search RSS, specific motifs known for mediation of DNA breaks (59 motifs) and interspersed and other types of repeats within particular DNA areas.

The uniformity test performed on breakpoint distribution within the minor (n=356) and Major (n=528) BCR areas revealed a non-random pattern (p=2.34e-21 and p=2.56e-10, respectively) with breakpoints accumulated in the second halves of the intron 1 (minor BCR) and intron 13 (Major BCR). Within the ABL1 area, breakpoint sites were distributed more randomly (p=9.28e-03); however, the breakpoint distribution within the ABL1 differed significantly between patients with CML and ALL (p=7.43e-05; see Figure) with higher accumulation of breakpoints near the 5’ end of the ABL1 intron 1 in CML and near the 3’ end of the intron 1 in ALL. The difference was not driven by the type of fusion (minor vs. Major BCR) as it was still visible even if only Major BCR::ABL1+ patients were analysed (414 CML and 108 ALL; p=2.28e-03). Comparison between various groups of patients (females vs. males; adults vs. children; various age groups, minor vs. Major BCR fusion [in ALL patients]), did not reveal a significantly distinct breakpoint distribution in any BCR or ABL1 area.

Analysis of genomic breakpoints showed that fusions are mostly formed in loci with short homologies (49%; 1–71bp, median=2bp), by blunt end junctions (36%) or by a junction with insertion of random nucleotides (13%; 1–42bp, median=3bp) strongly suggesting NHEJ mechanism of double-strand break repair in vast majority of cases. In 313 patients we compared BCR::ABL1 and the reciprocal ABL1::BCR fusion. Vast majority of the BCR::ABL1 and ABL1::BCR breakpoints (81% and 79% on BCR and ABL1 area, respectively) were located within ±100bp window, showing almost precise reciprocal translocation, with negligible losses/gains of DNA resulting from the fusion. Analysis of the association of breakpoints with DNA motifs and chromatin structure did not reveal any significant association with the localization of breakpoints.

In conclusion, we show different breakpoint distribution in ABL1 between CML and BCR::ABL1+ ALL. The double-strand breaks are repaired by NHEJ; however, we have not found any DNA or epigenetic motif, which might be responsible for the breakpoints. Our data suggest that the differences in breakpoint distribution are due to different chromatin accessibility and/or different relative positions of the BCR and ABL1 genes throughout the cell development, reflecting the origin of CML in earlier stages of hematopoiesis compared to ALL.

Supported by AZV (NU21-03-00128) and NICR No. LX22NPO5102.

Disclosures: Chiaretti: Pfizer: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees. Cario: Servier, Amgen: Research Funding; JazzPharma: Speakers Bureau.

*signifies non-member of ASH