Acute Myeloid Leukemia (AML) is a hematopoietic disease responsible for a large number of cancer-related deaths, with an estimated 10,600 deaths in 2017. Viruses are known causative agents for murine and avian myeloid leukemias; however, a viral etiology has not been definitively established for human AML. Identifying an infectious etiology may lead to an improved understanding of the disease which in turn leads to potential better diagnostics and therapeutic strategies. In this study, single-cell RNA sequencing was employed to analyse the transcriptomic landscape of mononuclear cells found in AML patients in an unbiased way. The sequencing data obtained were then mapped to a viral nucleotide database to identify potential viral RNA transcripts in the cells.

White blood cells or mononuclear cells were isolated from the bone marrow samples obtained from 3 AML patients by pre-staining with Hoescht, which selects for all white blood cells. Anti-CD34 antibody was also used to select for CD34 cells, which are known multipotent progenitor cells in the blood. Using the Automated Microfluidic C1 System, the isolated cells were lysed and the mRNA obtained was reverse transcribed and amplified. Using the Nextera XT DNA Sample preparation kit, cDNA library was prepared from the individual cells and processed on Illumina Next Sequencing (NexSeq) and HiSequencing 2000 (HiSeq) platform. Paired reads were generated for downstream analysis. The sequencing data were then mapped to a viral nucleotide database and mapped reads were analysed using an in-house virus identification script to determine the validity of the viral RNA transcripts found.

Single-Cell RNA sequencing was performed on 212 mononuclear cells, comprising 132 CD34 cells and 80 non-CD34 cells. The sequencing data obtained from 73 cells were successfully mapped to 13 different viruses. 44 of these cells observed to have viral RNA transcripts were CD34 cells. Some of the viruses that were found included Moloney Murine Leukemia Virus (M-MuLV), Murine Osteosarcoma Virus (MSV), Feline Sarcoma Virus (FeSV), Friend Murine Leukemia Virus (F-MuLV), Fujinami Sarcoma Virus (FJSV), Hepatitis C Virus (HCV), HERV-K Provirus (HERV-K), Adenovirus (ADV), Moloney Murine Sarcoma Virus (M-MuSV), Rauscher Murine Leukemia Virus (R-MuLV) and Tomato Mosaic Virus (ToMV).

Single-cell RNA sequencing was the method of choice as it enables us to analyze the transcriptomic landscapes at a single cell level, eliminating the likelihood of any minute viral RNA transcripts from being eclipsed as is the case with bulk sequencing. Through this, we have identified a variety of viral RNA sequences and it was observed that CD34 cells were found to harbor more viral RNA transcripts relative to non-CD34 cells. The presence of viruses in CD34 cells may cause genetic instability and lead to alteration of oncogenes or tumour-suppressor genes. These mutations in the progenitor cells could then be passed down to the cells it would differentiate to. However, further work is required to validate these observations and determine the properties of the identified viral sequences to be able to understand its role in tumorigenicity

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Disclosures

Chng: Janssen China R&D: Research Funding.

Topics:
leukemia, myelocytic, acute, rna, viral, cd34 antigens, sequence analysis, rna, nucleotides, antibodies, bone marrow specimen, dna, dna, complementary, leukemia, myeloid

Author notes

*

Asterisk with author names denotes non-ASH members.

© 2017 by The American Society of Hematology
2017
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