RNA-seq analysis of plasmatic exosomal miRNAs in pediatric Hodgkin Lymphoma

Introduction Hodgkin lymphoma (HL) represents around ~10% of all cases of lymphoid neoplasms. Although current risk-adapted and response based treatment approaches can cure >90% of the children, treatment of relapsed/resistant patients remains a challenge [1].

In the last years, a large body of evidence proved the functional involvement of exosomes in cancer progression and spreading, induction of angiogenesis, as well as in chemoresistance and immune response evasion during tumor development [2].

The aim of our study is to characterize miRNA in plasmatic exosomes of pediatric HL patients, for the identification of new non-invasive biomarkers and disease mechanisms, which might be used to improve diagnosis, treatment and prognosis of the affected children.

Methods The study cohort included 36 HL patients treated according to EuroNet-PHL-C2 trial and 7 plasma samples from healthy donors (D) as controls. Exosomes were isolated from 0.5 ml plasma at diagnosis by exoEasy Midi Kit (Qiagen) and validated by Nanoparticle Tracking Analysis (NTA, Malvern Nanosight), Atomic Force Microscopy (ATM) and Western Blotting. Total exosomal RNA was obtained using exoRNeasyMidi Kit (Qiagen) and sequencing libraries were prepared from 10 ng RNA by using the NEBNext® Multiplex Small RNA Library Prep Kit for Illumina® (New England Biolabs). RNA-seq analysis was performed on an Illumina HiSeq 4000 with target depth of 15 M reads per sample. RNA-seq data were analyzed using the software miR&moRe [3]. Differential expression analyses were performed with DESeq2 [4].

Results Overall, >200 expressed miRNAs were identified. Comparing HL and D exosome samples, 11 differentially expressed miRNAs were disclosed. When patients were grouped based on Early Response Assessment (ERA), 15 miRNAs differentially expressed were identified between patients with adequate and inadequate response.

Conclusion Differentially expressed miRNAs will be prioritized based on their functional significance in cancer and/or previous reports in lymphoid malignancies. The most promising candidates will be validated by quantitative real-time PCR in an extended cohort of both HL and D and functionally characterized by in vitro experiments.

These results are expected to provide insights on the role of exosomal miRNAs in HL disease aggressiveness and response to therapy.

• References

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