Blood-circulating EV-miRNAs, serum TARC, and quantitative FDG-PET features in classical Hodgkin lymphoma.

Blood-based biomarkers are gaining interest for response evaluation in classical Hodgkin lymphoma (cHL). However, it is unknown how blood-based biomarkers relate to quantitative 18F-FDG-PET features. We correlated extracellular vesicle-associated miRNAs (EV-miRNA), serum TARC, and complete blood count (CBC) with PET features (e.g., metabolic tumor volume [MTV], dissemination and intensity features) in 30 cHL patients at baseline. EV-miR127-3p, EV-miR24-3p, sTARC, and several CBC parameters showed weak to strong correlations with MTV and dissemination features, but not with intensity features. Two other EV-miRNAs only showed weak correlations with PET features. Therefore, blood-based biomarkers may be complementary to PET features, which warrants further exploration of combining these biomarkers in prognostic models.

Extracellular vesicle miRNA predict FDG-PET status in patients with classical Hodgkin Lymphoma.

Minimally-invasive tools to assess tumour presence and burden may improve clinical management. FDG-PET (metabolic) imaging is the current gold standard for interim response assessment in patients with classical Hodgkin Lymphoma (cHL), but this technique cannot be repeated frequently. Here we show that microRNAs (miRNA) associated with tumour-secreted extracellular vesicles (EVs) in the circulation of cHL patients may improve response assessment. Small RNA sequencing and qRT-PCR reveal that the relative abundance of cHL-expressed miRNAs, miR-127-3p, miR-155-5p, miR-21-5p, miR-24-3p and let-7a-5p is up to hundred-fold increased in plasma EVs of cHL patients pre-treatment when compared to complete metabolic responders (CMR). Notably, in partial responders (PR) or treatment-refractory cases (n = 10) the EV-miRNA levels remain elevated. In comparison, tumour specific copy number variations (CNV) were detected in cell-free DNA of 8 out of 10 newly diagnosed cHL patients but not in patients with PR. Combining EV-miR-127-3p and/or EV-let-7a-5p levels, with serum TARC (a validated protein cHL biomarker), increases the accuracy for predicting PET-status (n = 129) to an area under the curve of 0.93 (CI: 0.87-0.99), 93.5% sensitivity, 83.8/85.0% specificity and a negative predictive value of 96%. Thus the level of tumour-associated miRNAs in plasma EVs is predictive of metabolic tumour activity in cHL patients. Our findings suggest that plasma EV-miRNA are useful for detection of small residual lesions and may be applied as serial response prediction tool.

Plasma vesicle miRNAs for therapy response monitoring in Hodgkin lymphoma patients.

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).