Comprehensive Clinical and Molecular Characterization Reveals Rara As the Most Frequent Torque Teno Mini Virus Insertion Site and Defines a Distinct Ttmv::Rara Driven APL-like Leukemia Subtype
Authors: Sun Shu , Liu Yongjing , Xu Qing-Yu , Zhou Xiaosu , Wen Lijun , Chen Li , Chen Jiaqi , Wang Huili , Chen Xue , Lou Jiacheng , Zheng Huyong , Xie Yangyang , Wang Linya , Ma Futian , Yang Lihua , Wang Xianjing , Gao Wen , Wu Yin , Zhang Leping , Shen Shuhong , Chen Suning , Wang Kankan , Liu Hongxing , Huang Jinyan , Zhu Hong-Hu 
Published: December 2024

Torque teno mini virus (TTMV) is a ubiquitous human commensal virus that has been associated with leukemia and has the potential to integrate its sequence into the human genome. We developed a screening system to analyze TTMV integration in 8,608 leukemia patients using transcriptome data from public databases. Our algorithm identified 607 high-confidence TTMV insertion events, representing 412 unique gene-sample pairs (7%), with significant enrichment in the RARA gene (n=8) linked to an acute promyelocytic leukemia (APL) phenotype. TTMV::RARA was the most prominent fusion event among 3,604 acute myeloid leukemia (AML) samples. Although recent studies have reported cases of TTMV::RARA patients, a comprehensive characterization is still lacking. To address this knowledge gap, we collected a current largest dataset including clinical information and multi-omics data from 24 TTMV::RARA positive patients across 13 clinical centers and public databases, and conducted the first in-depth analysis of TTMV::RARA fusion events.

Consistent with previous reports, the spliced TTMV::RARA fusion gene typically features the TTMV sequence adjacent to the exon 3 of RARA, with possibly fewer than 10 intronic bases in between. Interestingly, we observed hotspots for TTMV insertion that were associated with specific RARA isoforms, suggesting potential pre-splicing integration events. Unsupervised clustering revealed that TTMV::RARA patients shared similar transcriptomic characteristics with APL. However, they exhibited a distinct clustering tendency. Further analysis identified two subclusters within the TTMV::RARA cohort, one of which had an remarkably high proportion (77.8%) of chromosome 17p deletions, a finding that is rare in leukemia (＜3% on average). Moreover, RARA gene expression was significantly higher in TTMV::RARA patients compared to other APL subtypes. Functional enrichment analysis further suggested an upregulation of energy metabolism activities in TTMV::RARA patients. Next, we compared the clinical and molecular biological features of TTMV::RARA cases with typical APL. Compared to typical APL, TTMV::RARA patients presented with a lower frequency of bleeding at diagnosis (21.7% vs 78%) and had higher platelet counts (median 83.8×109/L vs 28×109/L). Bone marrow morphology showed APL-like cells in both groups, but TTMV::RARA had a lower frequency of Auer rods (50% vs 94%). The proportion of high-risk patients(WBC&gt;10×109/L) was increased (47.6% vs 33%). Interestingly, 38% of TTMV::RARA patients also presented with myeloid sarcoma. These findings suggest that TTMV::RARA represents a distinct APL subtype with unique characteristics.

The developmental hierarchy of leukemic cells has been shown to influence disease phenotype and drug response, to investigate distinct tumor cell developmental stages in TTMV::RARA and typical APL, we compared single-cell transcriptome data from 3 TTMV::RARA patients and 5 PML::RARA patients. While GMP-like cells were the predominant leukemic population in both groups, TTMV::RARA harbored a higher proportion of cycling GMP-like cells, suggesting more active proliferation. The differentiated leukemic cells in TTMV::RARA were primarily Eosinophil-like, whereas those in PML::RARA were mainly Promono-like and Monocyte-like, indicating different differentiation trajectories.

Current clinical management of TTMV::RARA patients varies, and their responsiveness to all-trans retinoic acid (ATRA) is controversial. To facilitate the development of effective therapies, we retrospectively analyzed 20 TTMV::RARA patients with defined treatment regimens and complete follow-up information. TTMV::RARA exhibited a time-dependent response to ATRA +ATO (Arsenic trioxide) (CR: 62.5%, ＞14 days vs 16.7%, ≤14 days), and 84.6% of patients underwent hematopoietic stem cell transplantation (HSCT) with favorable outcomes. The 2-year cumulative incidence of relapse, event-free survival, and overall survival rates were 30.16%, 63.56%, and 83.9%, respectively. These findings suggest that prolonged ATRA+ATO treatment and HSCT may be beneficial for TTMV::RARA patients.

In conclusion, our study provides the first comprehensive landscape of TTMV insertions in AML and systematically characterizes the clinical features and multi-omics profiles of TTMV::RARA patients. We also reveal that TTMV::RARA represents a distinct APL subtype.