Chronic
lymphocytic leukemia (CLL) is an incurable disease, partly because leukaemic
cells resist the effects of conventional chemotherapy due to the contribution
to survival from the lymph node microenvironment. CLL cells communicate with
various types of normal cells of the microenvironment, such as mesenchymal
stromal cells, monocyte-derived nurse like cells (NLC) as well as T cells
(Caligaris-Cappio, Bertilaccio and Scielzo, 2014).
The
microenvironment is important in driving CLL proliferation, which occurs in
microanatomical tissue niches called proliferation centres
(pseudo-follicles), a key histopathological detecting in CLL (Duhren-von
Minden et al, 2012) Biomarkers derived specifically from CLL cells within the
microenvironment will be a direct measure of the numbers of these robustly
surviving cells and could improve treatment decisions, but such markers do
not exist. Importantly peripheral blood biomarkers derived from the lymph
node microenvironment will not only be clinically important in CLL but also
in the low-grade lymphomas, which are
relatively common diseases and for which clinical progression is
currently determined by clinical assessment and CT scans only.
Extracellular
vesicles (EVs) are produced by CLL cells only when they receive stimulation
such as cross-linking of the B-cell receptor or through CD40. EVs carry a
cargo, which includes miRNA. We reason that CLL cells generate EVs only when
receiving signals from cells in the lymph node microenvironment. In previous
work from the laboratory it has been shown that CLL cells stimulated by CD40L
and IL-4 produce EVs that are enriched in miR-363-3p and miR-374b. We
considered that these might be suitable biomarkers reflecting leukaemic
activity in the tumour microenvironment. Others showed that miR-363-3p and
miR-374b are not detectable at significant levels in plasma from normal
subjects and we, therefore, chose to pursue the hypothesis that plasma levels
of exosome miR-363 and miR-374b might correlate with disease activity within
lymph nodes (Arroyo et al, 2011).
The
numbers and sizes of EVs in plasma from patients with CLL (n=23) and normal
subjects (n=10) were measured using NanoSight dynamic light scattering
technology. Candidate miRNAs (miR-363-3p and miR-374b) were quantified in a
group of 25 patient plasma samples and 11 healthy volunteers utilizing
quantitative real-time PCR assays.
The
results show a significant difference in the number of particles between CLL
patients and normal subjects (Mann-Whitney U-test; P=0.03). Patients have
markedly increased numbers of these particles compared to healthy volunteers
(Mean; pts=8.744 x106/ml, HVs=4.620 x106/ml). Moreover, there is no
correlation between total white cell counts (WCC) or platelet count (PLT) and
EV numbers suggesting that EVs are only produced by CLL cells within lymph
nodes or bone marrow. The comparison of EV sizes between patients and normal
subjects shows no significant differences (Mean; pts=150 nm, HVs=155). In
addition, we observed that EV size distribution was not significantly
different between patients with high particle number and those with lower
numbers. QPCR tests for miR-363-3p have been carried out on patient plasma
and healthy volunteers and are extremely promising and suggest that it is
significantly elevated in patient groups compared with normal (MannWhitney
U-test; P=0.01). We are going to expand these assays to larger groups of
asymptomatic patients and patients who required treatment in order to
determine clinical usefulness. In addition, we are currently employing size
exclusion chromatography to identify the origin – plasma or extracellular
vesicles – of the miRNA in which we are interested.
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