Background:We have reported that peripheral blood cell-free DNA (cfDNA) is reliable for detecting bone marrow molecular abnormalities in patients with hematologic neoplasms. However, not clear is whether cfDNA is sufficient to detect mutations present at low variant allele frequency (VAF). Since patients with aplastic anemia (AA usually have relatively small clones in blood and bone marrow (BM), we compared mutations detected in BM cells with those detected in peripheral blood cfDNA from patientswith this disease.

Methods: A total of 120 paired BM aspirate and PB plasma samples were tested by the commercially available TruSight Myeloid Sequencing Panel (Illumina; San Diego, CA). We extracted DNA from bone marrow aspirate using the QIAamp DNA Mini Kit. We used NucliSenS EasyMAG automated platform for extracting total nucleic acid from PB plasma collected in EDTA. All paired BM and plasma samples were tested by the commercially available TruSight Myeloid Sequencing Panel (Illumina; San Diego, CA), which covers hot spot mutations in 54 genes. The average depth of sequencing was 10,000X.

Results: One hundred twenty paired BM and cfDNA samples from 96 patients with aplastic anemia were tested. Of the 96 patients, 33 (34%; equivalent to 48 samples, 40%) had one or more mutations. We identified 54 different somatic mutations in these patients, of which 45 were unique. There was no significant difference (P=0.71, Sign test) in allele frequency between cfDNA and BM. The median mutant allele frequency was 10.9% in cfDNA and 12.6% in BM cells, and 40 of the 54 mutations had allele frequency ≤20% in BM cells, while 45 samples had allele frequency ≤20 in cfDNA. Six of the 33 patients with somatic mutations (18%) showed mutations in plasma cfDNA but not in BM. In contrast, 2 patients (6%) showed mutations in BM cells and not in cfDNA. One of these two patients had a mutation in ASXL1 gene detected in BM cells but not in cfDNA and a subsequent sample showed the same ASXL1mutation in BM cells and not in cfDNA, and a second clone with a different ASXL1 mutation detected in both BM cells and cfDNA. Overall concordance between BM cells and cfDNA in the 120 samples was 92% and there was no statistically significant difference between the two sample types (P=0.6).

Summary and Conclusions: Seven samples (from 7 patients) of the 120 tested samples showed mutations in cfDNA and not in BM cells while 3 samples (from 2 patients) showed mutations in BM and not in cfDNA. VAF of mutations in cfDNA were similar to those in BM cells. Therefore, peripheral blood cfDNA should be tested in addition to BM cells for detecting mutations in patients with AA. Peripheral blood cfDNA can be used as a reliable means for monitoring patients with AA. cfDNA testing can be used as an alternative testing to bone marrow even when mutant allele frequency in bone marrow is <20%. cfDNA may be an especially valuable source of mutation detection in marrow failure, in which marrow aspirates may not contain sufficient cells for accurate mutation analysis.


Albitar:Neogenomics Laboratories: Employment. Townsley:Novartis: Research Funding. Ma:Neogenomics Laboratories: Employment. De Dios:Neogenomics Laboratories: Employment. Funari:Neogenomics Laboratories: Employment. Young:GSK/Novartis: Research Funding. Albitar:Neogenomics Laboratories: Employment, Equity Ownership.

Author notes


Asterisk with author names denotes non-ASH members.