Clonal evolution and clinical significance of copy number neutral loss of heterozygosity of chromosome arm 6p in acquired aplastic anemia.

Acquired aplastic anemia (aAA) results from the T cell-mediated autoimmune destruction of hematopoietic stem cells. Factors predicting response to immune suppression therapy (IST) or development of myelodysplastic syndrome (MDS) are beginning to be elucidated. Our recent data suggest most patients with aAA treated with IST develop clonal somatic genetic alterations in hematopoietic cells. One frequent acquired abnormality is copy-number neutral loss of heterozygosity on chromosome 6p (6p CN-LOH) involving the human leukocyte antigen (HLA) locus. We hypothesized that because 6p CN-LOH clones may arise from selective pressure to escape immune surveillance through deletion of HLA alleles, the development of 6p CN-LOH may affect response to IST. We used single nucleotide polymorphism array genotyping and targeted next-generation sequencing of HLA alleles to assess frequency of 6p CN-LOH, identity of HLA alleles lost through 6p CN-LOH, and impact of 6p CN-LOH on response to IST. 6p CN-LOH clones were present in 11.3% of patients, remained stable over time, and were not associated with development of MDS-defining cytogenetic abnormalities. Notably, no patient with 6p CN-LOH treated with IST achieved a complete response. In summary, clonal 6p CN-LOH in aAA defines a unique subgroup of patients that may provide insights into hematopoietic clonal evolution.

Emergence of clonal hematopoiesis in the majority of patients with acquired aplastic anemia.

Acquired aplastic anemia (aAA) is a nonmalignant disease caused by autoimmune destruction of early hematopoietic cells. Clonal hematopoiesis is a late complication, seen in 20-25% of older patients. We hypothesized that clonal hematopoiesis in aAA is a more general phenomenon, which can arise early in disease, even in younger patients. To evaluate clonal hematopoiesis in aAA, we used comparative whole exome sequencing of paired bone marrow and skin samples in 22 patients. We found somatic mutations in 16 patients (72.7%) with a median disease duration of 1 year; of these, 12 (66.7%) were patients with pediatric-onset aAA. Fifty-eight mutations in 51 unique genes were found primarily in pathways of immunity and transcriptional regulation. Most frequently mutated was PIGA, with seven mutations. Only two mutations were in genes recurrently mutated in myelodysplastic syndrome. Two patients had oligoclonal loss of the HLA alleles, linking immune escape to clone emergence. Two patients had activating mutations in key signaling pathways (STAT5B (p.N642H) and CAMK2G (p.T306M)). Our results suggest that clonal hematopoiesis in aAA is common, with two mechanisms emerging-immune escape and increased proliferation. Our findings expand conceptual understanding of this nonneoplastic blood disorder. Future prospective studies of clonal hematopoiesis in aAA will be critical for understanding outcomes and for designing personalized treatment strategies.

Persistence of anti-human leukocyte antibodies in congenital heart disease late after surgery using allografts and whole blood.

BACKGROUND: Allografts are used for vascular reconstruction in many forms of congenital heart disease. Although allografts induce anti-human leukocyte antibody (HLA) formation, much about this response is unknown. METHODS: Three groups of patients aged 8 to 18 years old underwent analysis for class I and II anti-HLA antibodies using Luminex. Groups were defined by timing of allograft exposure and diagnosis at Norwood for hypoplastic left heart syndrome (neonatal group), at Glenn for single-ventricle lesions not requiring arch reconstruction (infant group), and cardiac defects repaired during infancy without allografts (controls). Patients had significant anti-HLA (sensitization) if mean fluorescence intensity was ≥ 1500. RESULTS: The study enrolled 29 patients (median age, 10.1 years). Significant class I anti-HLA antibodies were seen in 44% (8 of 18) of the neonatal group, 25% (1 of 4) of the infant group, and 14% (1 of 7) of controls; class II anti-HLA antibodies were seen in 44% (8 of 18) of the neonatal group, 25% (1 of 4) of the infant group, and 29% (2 of 7) of controls. All patients received fresh whole blood, but the neonatal group had greater exposure (p = 0.001). There was less sensitization with increasing time from last receipt of allograft(s) or blood transfusion (p = 0.05). CONCLUSIONS: Exposure to allograft at the Norwood procedure is associated with long-term sensitization to anti-HLA antibodies in 56% of patients. Sensitization also occurs in those without prior exposure to allografts, may decrease over time, and appears related to whole blood. These findings have implications for those in whom heart transplant is considered late in the clinical course.

A strategy for single nucleotide polymorphism analysis of chimerism for somatic cell therapy.

BACKGROUND AIMS: Chimerism is an important outcome measure in hematopoietic cell transplantation as well as somatic cell therapy. Commonly used methods to estimate chimerism are restricted by either gender or inefficient sensitivity. In principle, real-time polymerase chain reaction (PCR)-based assays can be used to assess single nucleotide polymorphisms (SNP), which are a vast resource of molecular markers, and such assays demonstrate a substantially higher sensitivity (0.001%), but the specificity is unclear because of a low-level signal from mismatched sequences. METHODS: In this study, we cloned 14 pairs of SNP selected from the SNP HapMap database and examined the specificity and sensitivity of their detection by real-time PCR using two primer/fluorescent probe pairs to allow genotyping of the two possible variant alleles. Clinical donor-recipient pairs from 18 families were used to explore the efficacy of using SNP assays to measure chimerism. RESULTS: We found that the polymorphic nucleotide influences the ability to distinguish the signal generated by the target and mismatched sequences. Moreover, the specific fluorescent reporter probe can affect the difference in signal intensity between the target and mismatched sequences. Real-time PCR SNP assays can attain a sensitivity of 0.1-0.5% with 100% specificity. When comparing possible clinical donor-recipient pairs, we found an average 3.3 out of 14 SNP were informative. CONCLUSIONS: By optimal selection of the polymorphic sequences and fluorescent reporter, the real-time PCR SNP assay is superior to the short-tandem repeat chimerism assay and broadly applicable. This strategy may be applied in future clinical trials of bone marrow cell therapy.