Identification of Interstitial Loss of Heterozygosity (LOH) Regions Flanking CD33 Locus in AML Patients

Acute myeloid leukemia (AML) is a heterogenous disease with dismal overall survival of <30% in adults despite a >80% initial complete remission rates (CR). While standard intensive chemotherapy for AML consisting of cytarabine, daunorubicin and etoposide has remained unchanged for decades, addition of CD33-directed antibody-drug conjugate (ADC) gemtuzumab ozogamicin (GO; Mylotarg^TM) has shown decreased risk of relapse. CD33 is a cell surface antigen expressed in most AML blasts and internalizes upon binding to GO. However, polymorphisms within CD33 variants have been shown to abrogate this benefit of GO addition to chemotherapy. Our group has previously reported on the impact of 6 such polymorphisms on CD33 cell surface expression and association with clinical outcomes - rs12459419, rs2455069, rs35112940, rs61736475, rs1803254, and rs201074739. Among these, we have shown that the 4 bp indel rs201074739 results in premature termination codon and complete loss of CD33 expression in patients homozygous (del/del) for the deletion (PMID: 30721105).

Here, we present a preliminary report describing a novel chromosomal abnormality in the region containing and flanking the CD33 locus from 11 patients - AML (7), MDS (1), MPN (1), IgA gammopathy (1), or unknown (1). 8 patients presented as CD33 negative upon flow cytometric assessment and consisted of 4 in remission, 2 post-hematopoietic stem cell transplant (HCST) and 2 with unknown outcome. TaqMan genotyping revealed that 8 patients were homozygous for the rs201074739 CCGG deletion, while 3 were homozygous CCGG. Curiously, we observed that all 8 samples homozygous for rs201074739 del/del were also homozygous (either variant or wildtype allele) when genotyped for the other 5 CD33 SNPs listed above. In contrast, the samples homozygous for rs201074739 reference allele (CCGG/CCGG, no deletion) exhibited heterozygosity at other 5 SNPs to varying extent. This led us to hypothesize presence of a region of loss of heterozygosity (LOH) within or encompassing the CD33 locus in patients homozygous for rs201074739 CCGG deletion.

To test our hypothesis, we performed targeted next generation sequencing (NGS) using a custom panel (Twist Bioscience) designed to capture a ~224 kb region within chromosome 19q13.1 flanking the CD33 locus. Sequencing was performed using the Illumina NovaSeq X 2x150 platform to achieve a coverage of ~150x. Minimap2 was used for sequence alignment to hg38 and BCFtools for the subsequent variant calling. Integrative Genomics Viewer (IGV) was then used for visualization of the data.

Based on zygosity status of called variants, we observed 6 regions ranging from 5.9-23.6 kb with enrichment of LOH in all 8 rs201074739 CCGG del/del genotype samples but not in any CCGG/CCGG wildtype sample. Such tracts of LOH, ~10-30 kb in length, are termed interstitial LOH events and are reportedly distributed evenly within chromosomes unlike terminal LOH encompassing long stretches (>100 kb) mainly near telomeres. These 6 putative interstitial LOH regions mapped to the following genes: SIGLEC9, SIGLEC7, SIGLEC17p, SIGLEC1, CD33, and IGLON5.

We show for the first time interstitial LOH in chromosome 19q13.41 region linked with the presence of rs201074739 CCGG del/del in CD33 in patients with myeloid malignancies. The SIGLEC family of genes, including CD33, have been implicated in immune response functions and LOH in these could have implications beyond AML. IGLON5 is linked to an autoimmune neurodegenerative disorder, and LOH in this region is particularly interesting in the context of AML development in patients with such disorders. While interstitial LOH is reportedly the fundamental type observed across cancers, it occurs in solid tumors with a higher frequency compared to leukemias. However, interstitial LOH within chromosome 19q13 region specifically has been associated with aggressive forms of neuroblastoma and prostate cancer and occurs frequently in various gliomas. Furthermore, this could have implications for strategies utilizing CD33^-/donor cells generated for HSCT. In summary, our results add to and provide the potential to expand our understanding of the genomic complexity of AML, especially within myeloid marker CD33, a highly sought target in AML. Further studies will aim to test the biological and clinical relevance of these LOH regions flanking the CD33 locus.

Loken:Hematologics: Current Employment.