Plasma vesicle miRNAs for therapy response monitoring in Hodgkin lymphoma patients
Monique A.J. van Eijndhoven, Josée M. Zijlstra, Nils J. Groenewegen, Esther E.E. Drees, Stuart van Niele, S. Rubina Baglio, Danijela Koppers-Lalic, Hans van der Voorn, Sten F.W.M. Libregts, Marca H.M. Wauben, Renee X. de Menezes, Jan R.T. van Weering, Rienk Nieuwland, Lydia Visser, Anke van den Berg, Daphne de Jong, D. Michiel Pegtel
Monique A.J. van Eijndhoven, Josée M. Zijlstra, Nils J. Groenewegen, Esther E.E. Drees, Stuart van Niele, S. Rubina Baglio, Danijela Koppers-Lalic, Hans van der Voorn, Sten F.W.M. Libregts, Marca H.M. Wauben, Renee X. de Menezes, Jan R.T. van Weering, Rienk Nieuwland, Lydia Visser, Anke van den Berg, Daphne de Jong, D. Michiel Pegtel
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Clinical Research and Public Health Hematology Oncology

Plasma vesicle miRNAs for therapy response monitoring in Hodgkin lymphoma patients

Abstract

BACKGROUND. Cell-free circulating nucleic acids, including 22-nt microRNAs (miRNAs), represent noninvasive biomarkers for treatment response monitoring of cancer patients. While the majority of plasma miRNA is bound to proteins, a smaller, less well-characterized pool is associated with extracellular vesicles (EVs). Here, we addressed whether EV-associated miRNAs reflect metabolic disease in classical Hodgkin lymphoma (cHL) patients.

METHODS. With standardized size-exclusion chromatography (SEC), we isolated EV-associated extracellular RNA (exRNA) fractions and protein-bound miRNA from plasma of cHL patients and healthy subjects. We performed a comprehensive small RNA sequencing analysis and validation by TaqMan qRT-PCR for candidate discovery. Fluorodeoxyglucose-PET (FDG-PET) status before treatment, directly after treatment, and during long-term follow-up was compared directly with EV miRNA levels.

RESULTS. The plasma EV miRNA repertoire was more extensive compared with protein-bound miRNA that was heavily dominated by a few abundant miRNA species and was less informative of disease status. Purified EV fractions of untreated cHL patients and tumor EVs had enriched levels of miR24-3p, miR127-3p, miR21-5p, miR155-5p, and let7a-5p compared with EV fractions from healthy subjects and disease controls. Serial monitoring of EV miRNA levels in patients before treatment, directly after treatment, and during long-term follow-up revealed robust, stable decreases in miRNA levels matching a complete metabolic response, as observed with FDG-PET. Importantly, EV miRNA levels rose again in relapse patients.

CONCLUSION. We conclude that cHL-related miRNA levels in circulating EVs reflect the presence of vital tumor tissue and are suitable for therapy response and relapse monitoring in individual cHL patients.

FUNDING. Cancer Center Amsterdam Foundation (CCA-2013), Dutch Cancer Society (KWF-5510), Technology Foundation STW (STW Perspectief CANCER-ID).

Authors

Monique A.J. van Eijndhoven, Josée M. Zijlstra, Nils J. Groenewegen, Esther E.E. Drees, Stuart van Niele, S. Rubina Baglio, Danijela Koppers-Lalic, Hans van der Voorn, Sten F.W.M. Libregts, Marca H.M. Wauben, Renee X. de Menezes, Jan R.T. van Weering, Rienk Nieuwland, Lydia Visser, Anke van den Berg, Daphne de Jong, D. Michiel Pegtel

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Figure 2

Small RNA distribution and recovery in EV fractions 9 and 10.

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Small RNA distribution and recovery in EV fractions 9 and 10. (A and B) ...
(A and B) RNA distribution of miR142-3p, let7a-5p, and vtRNA1-1 (A) and miR92a-3p, miR21-5p, and miR451-5p (B) in 26 fractions upon size-exclusion chromatography (SEC) of 1.5 ml healthy donor plasma. Total RNA was isolated with TRIzol followed by RT-PCR. Data are depicted as raw Ct values; error bars represent SEM from PCR duplicates. (C) Fold enrichment of vtRNA1-1, let7a-5p, and miR142-3p in plasma extracellular vesicles (EVs) (fractions 9 and 10) compared with protein/HDL (fractions 20 and 21). Data are shown as the mean of 2 donors; dots indicate individual samples. (D) Fold enrichment of miR92a-3p, miR21-5p, and miR451-5p in protein/HDL (fractions 20 and 21) compared with plasma EVs (fractions 9 and 10). Data are shown as the mean of 2 donors; dots indicate individual samples. (E) Fold enrichment of vtRNA1-1 in tumor EV (tEV; fractions 9 and 10) compared with protein/HDL (fractions 20 and 21) after SEC of 1.5 ml B cell culture supernatant. (F) SEC of 1.5 ml healthy donor plasma after spike in with 50 μl tumor cell line–derived exosomes. Shown is the fold increase of EBV-miR BHRF1-3 and BART2-5p in EV (fractions 9 and 10) compared with protein/HDL (fractions 20 and 21). Data are shown as the mean of the 2 consecutive SEC fractions; dots represent individual fractions (E and F).