Single Nucleotide Polymorphism Array Analysis Of Bone Marrow Failure Patients Reveals Characteristic Patterns Of Genetic Changes

Abstract

Bone marrow failure syndromes (BMFS) are a diverse group of rare life-threatening blood disorders characterized by inadequate hematopoiesis, clonal evolution, and increased risk of hematologic malignancies. Despite recent advances in the understanding of the molecular pathogenesis of BMFS, the ability to diagnose, risk-stratify, and treat patients with these rare disorders remains limited. In both the acquired and the inherited BMFS, the major contributors to mortality are complications of progressive cytopenias, and, albeit to a lesser extent—transformation to myelodysplastic syndrome (MDS) and acute myeloid leukemia. The main predictor of malignant transformation is acquisition of clonal cytogenetic abnormalities. Recently, single nucleotide polymorphism arrays (SNP-A) were proposed as a promising tool for high resolution cytogenetic analysis and surveillance of early clonal changes in BMFS, however, their clinical utility still remains to be established. In 2009, the Comprehensive Bone Marrow Failure Center at the Children’s Hospital of Philadelphia and the Hospital of the University of Pennsylvania incorporated high-density SNP-A as an adjunct to conventional cytogenetics in evaluation of BMFS patients. Here we present a comprehensive analysis of genetic changes in BMFS using 124 SNP-A from 91 patients, who were referred for evaluation of bone marrow failure. SNP-A genotyping was correlated with medical histories, hematopathology, cytogenetic, and molecular data. To assess the potential role of SNP-A in screening for early clonal evolution, longitudinal analysis of SNP-A was performed in 25 patients. We found that acquired copy number-neutral loss of heterozygosity (CN-LOH) was significantly more frequent in acquired aplastic anemia (aAA) than in other BMFS (OR 12.240, 95% CI 1.333-573.696, p<0.01). In contrast, acquired copy number alterations (CNAs) were more typical of MDS and unclassified BMFS. Extended tracts of homozygosity were common, frequently unmasking recessive loci in cases of inherited BMFS. Copy number variants (CNVs) were frequently polymorphic, and we identified several CNVs that are enriched in patients with aAA and neutropenia and may serve as disease modifiers. Clinical utilization analysis revealed that SNP-A can be helpful as an adjunct to conventional cytogenetics at the time of initial diagnosis (e.g. to identify regions of acquired CN-LOH and inherited homozygosity, acquired CNAs with a small clone size, and CNVs). Our longitudinal analysis showed that the likelihood of detecting a new acquired abnormality in a follow-up SNP-A was significantly higher in the setting of relapse than in the setting of stable disease (OR 27, 95% CI 1.23 to 808.54, p=0.035). Our results suggest that acquired CN-LOH is a general phenomenon in aAA, likely mechanistically and prognostically distinct from typical CN-LOH of myeloid malignancies. Our analysis of clinical utility of SNP-A shows the highest yield of detecting new clonal hematopoiesis at diagnosis and at relapse. Disclosures: No relevant conflicts of interest to declare.

Assessing copy number aberrations and copy neutral loss of heterozygosity across the genome as best practice: An evidence based review of clinical utility from the cancer genomics consortium (CGC) working group for myelodysplastic syndrome, myelodysplastic/myeloproliferative and myeloproliferative neoplasms

“…Serial longitudinal CMA analyses were able to identify monosomy 7 in four AA patients, consistent with progression, earlier than karyotype analysis [77]. CN-LOH of 6p involving the HLA locus was a frequent abnormality seen in 11% of AA patients [78,79]. CN-LOH of 6p is a mechanism for cells evading the immune system, rather than a malignant process and is often associated with multiple clones.…”

Acquired Aplastic Anemia as a Clonal Disorder of Hematopoietic Stem Cells

“…One of these alterations is copy numberthe neutral loss of heterozygosity of chromosome arm 6 p (6p CN-LOH), which is the major histocompatibility complex (MHC) region reach in the multiple human leucocyte antigen (HLA) loci. The 6p CN-LOH is more frequent in AA compared to other bone marrow failure syndrome [29]. The 6p CN-LOH was detected in only 13% of AA in the Japanese population [30].…”

“…In 25% of AA patients, progression into clinical hemolysis after immunosuppressive therapy is observed after clone size raises up to 37% for erythrocytes and 28% for granulocytes [101]. Babushok et al identified two potential driver mutations in HLA alleles, STAT5B and CAMK2G, associated with translational consequences in pathways of hematopoietic growth and immunity [29].…”

Clonal Evolution and Clinical Significance of Copy Number Neutral Loss of Heterozygosity of Chromosome Arm 6p in Acquired Aplastic Anemia

“…[6] Recent studies have identified acquired copy number neutral loss of heterozygosity on the short arm of chromosome 6 (6p CN-LOH) as a recurrent clonal abnormality uniquely present in aAA but interestingly not in inherited bone marrow failure conditions. [5][6][7][8][9] The HLA loci map to chromosome 6p, and deletions or LOH in this region result in the loss of alleles from one parental HLA haplotype within the affected clone, suggesting that 6p CN-LOH may emerge as the result of selective pressure to escape the auto-reactive immune response in aAA. [6] We hypothesized that the development of 6p CN-LOH hematopoietic clones defines a unique subgroup of patients with aAA who may have a distinct response profile to IST and are likely to exhibit other features of immune-driven clonal evolution.…”