Abstract
Gene abnormalities, including mutations and fusions, are important determinants in the molecular diagnosis of myeloid neoplasms. The use of bone marrow (BM) smears as a source of DNA and RNA for next-generation sequencing (NGS) enables molecular diagnosis to be done with small amounts of bone marrow and is especially useful for patients without stocked cells, DNA or RNA. The present study aimed to analyze the quality of DNA and RNA derived from smear samples and the utility of NGS for diagnosing myeloid neoplasms. Targeted DNA sequencing using paired BM cells and smears yielded sequencing data of adequate quality for variant calling. The detected variants were analyzed using the bioinformatics approach to detect mutations reliably and increase sensitivity. Noise deriving from variants with extremely low variant allele frequency (VAF) was detected in smear sample data and removed by filtering. Consequently, various driver gene mutations were detected across a wide range of allele frequencies in patients with myeloid neoplasms. Moreover, targeted RNA sequencing successfully detected fusion genes using smear-derived, very low-quality RNA, even in a patient with a normal karyotype. These findings demonstrated that smear samples can be used for clinical molecular diagnosis with adequate noise-reduction methods even if the DNA and RNA quality is inferior.
Highlights
Gene mutations are essential prognostic factors in diagnosing and predicting the effect of therapy on myeloid neoplasms [1, 2]
While the analysis showed that smear slides for Next-generation sequencing (NGS) can be used to create gene mutation profiles, it is still unclear whether they can provide insight into other myeloid malignancies, information about the deterioration of data, including gene-expression noise in smear samples, and the utility of RNA derived from this source
The dsDNA/total DNA ratio in each sample indicating the degree of DNA decay was significantly lower (P = 0.0079) in the smear samples than in the bone marrow cell (BMC) (Fig 1A)
Summary
Gene mutations are essential prognostic factors in diagnosing and predicting the effect of therapy on myeloid neoplasms [1, 2]. Previous studies examined the utility of slides containing biopsy samples as a source of DNA and RNA for target sequencing of lung adenocarcinoma [5] and thyroid cancer [6] and were able to provide profiles of gene mutations, including driver and drug-resistance mutations, suggesting that preserved or pretest samples can be used for NGS. In these cases, the samples were prepared using formalin-fixed, paraffinembedded (FFPE) slides that allow preservation for extended periods of time unlike BM aspirate smears made by drying and alcohol-based fixation. The present study analyzed the quality of DNA and RNA in BM smear samples and assessed their utility in NGS analysis by analyzing the character of the variants detected
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.