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Objective: 3′ tRNA‐derived fragments (3′ tRFs) are important epigenetic regulators in normal and pathological conditions. In this study, we aimed to explore the potential value of a 3′ tRF as a prognostic and/or screening biomarker for B‐cell chronic lymphocytic leukemia (B‐CLL). Methods: Publicly available next‐generation sequencing data from 20 B‐CLL cases were analyzed, followed by prediction of targets of the most abundantly and ubiquitously expressed 3′ tRFs, leading to selection of tRF‐LeuAAG/TAG. PBMCs were isolated from blood samples of 91 B‐CLL patients and 43 non‐leukemic donors, followed by total RNA extraction, in‐vitro polyadenylation, and first‐strand cDNA synthesis. Next, a real‐time quantitative PCR (qPCR) assay was developed for the accurate quantification of tRF‐LeuAAG/TAG and applied in all samples, prior to biostatistical analysis. Results: High tRF‐LeuAAG/TAG levels are associated with inferior overall survival (OS) of B‐CLL patients. The unfavorable significance of tRF‐LeuAAG/TAG was independent of established prognostic factors in B‐CLL. Stratified Kaplan‐Meier OS analysis uncovered the unfavorable prognostic role of high tRF‐LeuAAG/TAG levels for patients in Binet A or Rai I stage, negative CD38 expression, mutated, or unmutated IGHV genomic locus. Conclusion: Our approach revealed the independent prognostic value of a particular 3′ tRF, derived from tRNALeuAAG and tRNALeuTAG (tRF‐LeuAAG/TAG) in B‐CLL. [ABSTRACT FROM AUTHOR]

• A qPCR assay was developed for the quantification of a new mt-tRNA-derived fragment. • i-tRF-PheGAA can distinguish between CLL patients and normal controls. • An association was observed between i-tRF-PheGAA expression and CLL clinical stage. • Patients overexpressing i-tRF-PheGAA show significantly poorer overall survival. • The prognostic power of i-tRF-PheGAA is independent of established prognosticators. Chronic lymphocytic leukemia (CLL) is the most common leukemia among adults. The prognosis of CLL patients varies considerably. Transfer RNA-derived RNA fragments (tRFs) constitute a class of small non-coding RNA fragments excised from mature tRNAs and pre-tRNAs located in nuclei as well as in mitochondria. In this study, the clinical utility of i-tRF-PheGAA, a novel mitochondrial tRF, was investigated in CLL. Peripheral blood mononuclear cells (PBMCs) were isolated from 91 CLL patients and 43 non-leukemic controls. Total RNA was isolated from each sample, polyadenylated at the 3′ end and reversely transcribed. An in-house developed real-time quantitative PCR assay was developed and applied, and the results were biostatistically analyzed. For the normalization of the i-tRF-PheGAA expression levels, the expression of a small nucleolar RNA (RNU48) was used as reference. Mann-Whitney U test showed that i-tRF-PheGAA can distinguish between CLL samples and normal controls (p < 0.001). As determined by Kaplan-Meier survival analysis, overexpression of i-tRF-PheGAA was related to poor overall survival of the CLL patients (p < 0.001). Univariate bootstrap Cox regression analysis exhibited a higher hazard ratio of 7.95 (95% CI = 2.37–26.72, p < 0.001) for patients with positive i-tRF-PheGAA expression status. Multivariate bootstrap Cox regression analysis showed that the prognostic value of this tRF is independent of clinical stage, mutational status of the immunoglobulin heavy chain variable (IGHV) genetic locus, and CD38 expression status (p = 0.010). Our results show that i-tRF-PheGAA can serve as a molecular biomarker of poor prognosis in CLL, alongside with the existing factors for CLL prognosis. [ABSTRACT FROM AUTHOR]

• An innovative qPCR method for quantification of i-tRF-GlyCCC levels was developed. • High i-tRF-GlyCCC expression predicts poor overall survival in CLL patients. • Prognostic significance of i-tRF-GlyCCC is independent of other prognostic factors. • i-tRF-GlyCCC is an unfavorable prognosticator in distinct subgroups of CLL patients Chronic lymphocytic leukemia (CLL) is one of the most common types of leukemia in adults. Several studies have identified various prognostic biomarkers in CLL. In this study, we investigated the potential value of an internal fragment of the tRNAs bearing the Glycine anticodon CCC (i-tRF-GlyCCC), which is a small non-coding RNA, as a prognostic and screening biomarker in CLL. For this purpose, blood samples were collected from 90 CLL patients and 43 non-leukemic blood donors. Peripheral blood mononuclear cells (PBMCs) were isolated, total RNA was extracted and in-vitro polyadenylated, and first-strand cDNA was synthesized using an oligo-dT–adaptor primer. A real-time quantitative PCR assay was developed and applied for the quantification of i-tRF-GlyCCC in our samples. The biostatistical analysis revealed that i-tRF-GlyCCC levels are significantly lower in PBMCs of CLL patients, compared to PBMCs of non-leukemic controls, and that i-tRF-GlyCCC could be considered as a screening biomarker. Kaplan-Meier overall survival (OS) analysis revealed reduced OS for CLL patients with positive i-tRF-GlyCCC expression (P = 0.001). Multivariate Cox regression confirmed its independent unfavorable prognostic power with regard to OS. In conclusion, i-tRF-GlyCCC may constitute a promising molecular biomarker in CLL, for screening and prognostic purposes. [ABSTRACT FROM AUTHOR]