Materials and methods
Patient samples
Plasma samples from normal subjects (n = 11) were obtained after informed consent was obtained (Leicester Research Ethics Committee 06/Q2501/122). Plasma samples were obtained through the UK CLL Trials Biobank (University of Liverpool) (North-West England Research Ethics Committee 14/NW/1014) from CLL patients enrolled in two clinical trials: the ARCTIC trial which was funded by the NIHR Health Technology Assessment Programme (NIHR HTA project number 07/01/38; ISRCTN16544962) (University of Leeds) [19] (n = 100) and CLEAR [A trial looking at using antibiotics for chronic lymphocytic leukaemia (http://www.cancerresearchuk.org/about-cancer/find-a-clinical-trial/a-trial-looking-using-antibiotics-for-chronic-lymphocytic-leukaemia-the-clear-trial)] (n = 50). For ARCTIC, a trial investigating advanced disease requiring treatment, median age was 63 years, interquartile range 58–67 years and M:F was 69:31. 48 patients had unmutated immunoglobulin genes, 36 mutated and 16 not determined. 14 patients showed 11q23 deletion and 4 patients 17p deletion by FISH interphase cytogenetics. Clinical information has not yet become available for patients enrolled in CLEAR, a trial enrolling asymptomatic patients with early stage disease. It was not possible to complete processing of 5 ARCTIC samples and 2 CLEAR samples, either because miRNA isolation failed or RT-PCR failed, and these cases were, therefore, excluded from the study.
Size exclusion chromatography
An ÄKTA Prime (GE Healthcare, Little Chalfont, UK) with a sephacryl S-500 resin chromatography column (0.9 × 30 cm, 19.1 ml bed volume) was employed to fractionate plasma samples. Before injection, the column was equilibrated with phosphate buffered saline (PBS) (pH 7.4) (25 ml) solution at 0.5 ml/min at room temperature. Platelets were depleted from fresh plasma by two rounds of centrifugation. The sephacryl column was then injected with 7 ml of undiluted plasma and eluted at room temperature for approximately 1 h with PBS solution at a flow rate of 0.5 ml/min. A total of 31–37 fractions of 4 ml each were collected. The column was flushed with 75 ml of PBS solution at 0.5 ml/min (3.75 column volumes) between plasma fractionation to eliminate carryover. Protein molecular weight standard BSA (67 kDa; GE Healthcare) was used. Fractions were stored at 4 °C before use.
MiRNA isolation and quantitative RT-PCR
QIAzol Lysis Reagent (Qiagen, Hilden, Germany, Cat No. 79306) was added to ultra-centrifuged plasma samples. The upper, aqueous phase was extracted, and ethanol was added to provide appropriate binding conditions for all RNA molecules from approximately 18 nucleotides (nt) upwards. The sample was then applied to the RNeasy MinElute spin column (MiRNeasy Kit, Qiagen, Cat No. 217004) and RNA eluted in RNase-free water.
To assess recovery and stability of RNA, each sample was spiked with an identical amount of synthetic UniSp2 RNA (Exiqon, Vedbaek, Denmark, #203203). Patient samples were taken into heparinised tubes but heparin is an inhibitor of enzymatic reactions. Therefore, plasma samples were treated with heparinase I (H2519; Sigma, St. Louis, MO, USA) according to the manufacturer’s instructions. Extracted miRNA were then reverse transcribed using Universal cDNA synthesis kit (Exiqon, #203301) according to the manufacturer’s protocol. The template RNA samples were diluted to a concentration of 40 ng/µl using nuclease free water. The reaction was incubated for 60 min at 42 °C before termination.
Standard curves (Fig. 1) were constructed using synthetic oligonucleotide templates (Sigma, St. Louis, MO, USA) for miR-16 (UAGCAGCACGUAAAUAUUGGCG), miR-363-3p (5′-AAUUGCACGGUAUCCAUCUGUA), miR-142-3p (5′-UGUAGUGUUUCCUACUUUAUGGA) and let-7a-5p (5′-UGAGGUAGUAGGUUGUAUAGUU) (ThermoFisher, Waltham, MA, USA).
Quantitative PCR reactions were performed using SYBR green and miRNA-specific primers (Exiqon, hsa-miR-363-3p LNA PCR primer set #204726, hsa-miR-142-3p LNA PCR primer set #204291, hsa-let-7a-5p LNA PCR primer set #206084, hsa-miR-16-5p LNA PCR primer set #205702) according to the manufacturer’s instructions. cDNA produced in the RT reaction was amplified in MicroAmpTM optical 96-well reaction plates in triplicate 10 µl reactions on an Applied Biosystems 7900HT Thermocycler. Concentration and quality of nucleic acids were checked using NanoDrop® ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington DE, USA).
Results
Plasma MiR-363 levels are elevated in patients as compared to normal subjects
Levels of miR-363 were compared between normal subjects (n = 11) and CLL patients with early stage (CLEAR) (n = 48) or advanced (ARCTIC) disease (n = 95). Levels in patients vary over ~ 1000-fold, from 104 to 107 copies/µl, and were significantly higher in patients with advanced disease as compared to patients with early stage disease (P = 0.0091, Mann–Whitney test) or normal subjects (P = 0.0313) (Fig. 2a) while there was no significant difference between normal subjects and patients with early stage disease. Immunoglobulin gene mutational status is an established CLL prognostic marker but for patients with advanced disease there was no significant difference in miR-363 levels between those with mutated or unmutated immunoglobulin genes (Fig. 2b). Cytogenetic aberrations are associated with clinical outcome. Deletion of 17p and 11q23 are both associated with reduced overall survival as compared to patients with normal karyotypes [20]. Analysis of ARCTIC data showed there was no significant difference in miR-363 levels in patients with either 17p deletion (n = 4) or 11q23 (n = 14) deletion as compared to those with a normal karyotype. Similarly neither gender nor Binet clinical stage were associated with significant differences in amounts of miR-363 (Fig. 2c, d).
We compared outcomes for patients with miR-363 levels greater than the median for the group with those whose miR-363 level was less than that of the median (Fig. 2e, f). There was no significant difference between these groups in either overall or progression free survival.
Therefore, miR-363 levels are higher in patients with advanced disease but there is no association between higher levels and prognostic markers or clinical outcome.
Circulating miR-363 distribution between plasma protein and particle bound fractions
Others have shown that, in single donors, miR-16 and miR-363 co-fractionate with plasma protein fractions whereas let-7a and miR-142 co-fractionate with large protein complex/particle fractions [1]. We wished to investigate miR-363 because of our previous work including having established that there are higher circulating levels in patients as compared to normal subjects. Based on the work of Arroyo et al. [1] miR-16 acted as a control for plasma protein bound miRNA whereas miR-142 and let-7a were controls for more particle bound miRNA. Total amounts of let-7a and miR-142 (0.8 × 104 ± 0.3 × 104 and 0.3 × 104 ± 0.08 × 104 copies/µl respectively, mean ± SEM) were much lower than for miR-16 (4.1 × 104 ± 2.6 × 104 copies/µl). In normal subjects miR-363 was highly expressed (10.6 × 104 ± 4.6 × 104 copies/µl) and was detectable in the small protein fractions (97% in fractions 13–31) (Fig. 3). As expected there were greater amounts of total miRNA in patients: miR-16 (30.5 × 104 ± 14.8 × 104 copies/µl) and miR-363 (55.0 × 104 ± 23.7 × 104 copies/µl) and to a lesser extent for miR-142 (1.8 × 104 ± 0.8 × 104 copies/µl) and let7a (1.3 × 104 ± 0.4 × 104 copies/µl).
Increased levels of miR-363 due to EV release by activated CLL cells in the TME might lead to an increased proportion of circulating and particle bound miRNA. In order to determine the distribution of miR-363 in patients (n = 4) and normal subjects (n = 3) between particle bound and plasma protein fractions we carried out size exclusion chromatography followed by quantitative RT-PCR.
We confirmed that in normal subjects miR-16 co-fractionated with the plasma protein fractions (96% in fractions 14–31) (Fig. 3) but let-7a and miR-142 were distributed more evenly across particle bound and plasma protein fractions (50 and 56% respectively in fractions 1–13). There were significant differences in distribution in patients as compared to normal subjects. Patients showed relatively more miRNA in the early eluting large protein/particle fractions than normal subjects. For miR-16 21% was in fractions 1–13, which is significantly more than in the later fractions (P = 0.0061, Mann–Whitney test) and similarly for miR-363 23% was present in the early fractions, which was again significantly more than in the later fractions (P = 0.033). These differences were not observed for miR-142 or let-7a.
Discussion
There is a wealth of data to show that the tumour microenvironment (TME), which for CLL can be either lymph node or bone marrow, is essential for driving leukemic cell proliferation and mediates survival in the face of chemotherapy. By contrast circulating leukemic cells are predominantly non-dividing and quiescent. A reasonable hypothesis is that a marker of leukemic cell activity in the TME will be useful in guiding clinical decisions.
Our focus has been on miRNA, oligonucleotides with essential roles in regulating gene expression [21]. MiRNA are part of the cargo of EVs and we [12] and others [22] have proposed that they mediate some aspects of intercellular communication in the TME. MiRNA are also readily detectable in the blood and patterns of miRNA can be diagnostic for specific cancers [23] including CLL [11]. Circulating miRNA also hold promise as predictive biomarkers in cancer [24, 25] and in CLL [10].
Others have investigated the distribution of specific miRNAs between plasma protein and particle bound fractions [1]. In the normal subjects that these authors investigated the majority of miRNA were found in the plasma protein and not the particle fractions and they demonstrate that Argonaute2 co-purifies and stabilises these miRNA. A minority of miRNA, in normal subjects, associated with particles.
We focused on miR-363 because our work suggested enrichment in EVs following stimulation of leukemic cells by CD40L/IL-4 [26] and that particle bound miR-363 perturbs several functions of autologous CD4+ T-cells in vitro [12]. Others showed, in a small number of patients, that circulating miR-363 levels associated with clinical stage of CLL [10]. In normal subjects miR-363 appears to belong to the majority of miRNA that are predominantly in plasma protein fractions.
Our study is the first to investigate changes in the distribution of miRNA between protein and particle fractions of plasma in a disease. Four miRNA were investigated miR-363, miR-142, miR-16 and let-7a. In addition to miR-363, miR-16 is elevated in the plasma of CLL patients as compared to normal subjects (or patients with myeloma or hairy cell leukemia) [10] and might have a function in the development of this condition [27]. Like miR-363 in normal subjects miR-16 is predominantly in plasma protein fractions [1] but let-7a and miR-142 are mostly present in the particle fractions. In our group of normal subjects we demonstrate presence of the majority of miR-363 and miR-16 in plasma protein fractions confirming the previous work [1]. We did not find a clear separation of miR-142 or let-7a between plasma protein and particle fractions in normal subjects, although at least 50% was present in particle bound fractions. However, patients show a clear increase in levels of particle bound miR-363 and miR-16, which is not observed for miR-142 or let-7a.
We speculate that enrichment of miR-363 and miR-16 in EVs from CLL cells in the TME is reflected in increased circulating and particle bound miRNA in patients as compared to normal subjects. This represents a new parameter for defining differences between normal subjects and patients and it will be interesting to discover if this principle applies to other cancers.