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progression such as serum alkaline phosphatase level (<30
vs 130 U/l), lactate hydrogenase (<250 vs 250 U/l), and
hemoglobin (Hb) levels (<12 vs 12 g/dl). This latter was
the only one that correlated with a better PFS (25 mo vs
3 mo; p < 0.05). Owing to the small sample size and the
limited number of events, the multivariable model included
only AR-V7 status, AR-V7 expression levels, prior use of
taxanes, and Hb levels. However, probably due to the small
population, the multivariable model did not confirm the
role of AR-V7 as an independent prognostic factor (hazard
ratio: 3.6; p = 0.18).
4. Discussion
Vesicles released by cancer cells are loaded with nucleic
acids and proteins and represent tumor heteroge-
neity. As an alternative, CTCs may be used to interrogate
tumor biology however, the extraction and
processing of exosomes is considerably less expensive
and labor intensive than CTCs. Additional limitations of the
use of CTCs are morphologic and immune-phenotypic
heterogeneity (ie, epithelial markers may significantly vary)
and the fragility of CTCs, causing inaccurate detection
of AR-V7. The comparison of CTCs versus plasma-
derived exosomes as a source of RNA (present study)
demonstrates that CTC AR-V7+
[2_TD$DIFF] patients have a median OS of
8 mo versus not reached for AR-V7 patients a result
similar to the present study (8 mo vs not reached,
respectively, AR-V7+ vs AR-V7 patients). However, PFS
of patients stratified as AR-V7+ versus AR-V7 by the two
methods was different: 2.2 versus 6.2 mo for CTCs and
3 versus 20 mo for exosomes. The higher PFS of AR-V7
patients (exosomal RNA as a source) may depend on false
negatives in the cohort of participants whose RNA was
extracted from CTCs, thus reflecting a higher sensitivity of
the exosome approach over CTC. RNA for AR-V7 analysis
may also be extracted from whole blood but this
approachmay be adversely affected by the instability of free
RNA in blood and the large amount of contaminating RNA
from leucocytes. This is possibly the reason why the
analysis of AR-V7 mRNA in whole blood failed to predict the
PSA response/resistance in patients treated with HT
However, comparisons between methods performed
on different samples should be made with great caution.
Although promising, the clinical role of the AR-V7 as a
predictive biomarker of resistance to HT in CRPC and its
inclusion in clinical practice is still being debated because of
the small number of enrolled patients in published studies.
Several reports demonstrated and validated its role not only
in vitro, but also in vivo The studies on CTCs
, tissue samples from biopsies and our study on
exosomal RNA clearly confirm that AR-V7+ patients have
both lower PSA RR and shorter PSA PFS compared with AR-
V7 participants. In a recent study on AR-V7 analysis on
CTCs, it was shown that the incidence and amount of AR-V7
increase by line of therapy, reconfirming the role of AR-V7
as an acquired mechanism of resistance to systemic therapy
. An immunohistochemical assay was recently validat-
ed for AR-V7 detection in tissue biopsy . The study
demonstrated a clear effect of AR-V7 on survival parameters
and confirmed the predictive role of this biomarker on OS in
patients stratified in tertiles depending on AR-V7 (15.6 vs
9.6 vs 8.8 mo, respectively) However, the use of this
approach to monitor the development of resistance is
limited by the invasiveness of the procedure. In addition, a
single tissue sample is not representative of tumor
heterogeneity, and molecular evolution over time cannot
be monitored, making this approach scientifically sound but
practically less attractive. This study first demonstrates that
plasma-derived exosomes provide a viable source of RNA
for AR-V7 analysis and confirms its role as a strong
biomarker of resistance to HT in CRPC patients.
Although in previous studies AR-V7 levels
increased over time, in our study only four of seven
samples had increasing AR-V7 copies/ml. An explanation is
that additional mechanisms of resistance may have been
developed.
5. Conclusions
The present study provides evidence that RNA recovered
from plasma-derived exosomes may offer obvious advan-
tages, streamlining the diagnostic workflow by reducing the
costs, eliminating the invasiveness of tissue sampling, and
minimizing the negative effect of tumor heterogeneity.
Author contributions: Marzia Del Re had full access to all the data in the
study and takes responsibility for the integrity of the data and the
accuracy of the data analysis.
Study concept and design: Del Re, Danesi.
Acquisition of data: Del Re, Biasco, Derosa, Galli.
Analysis and interpretation of data: Del Re, Biasco, Rofi, Crucitta, Derosa,
Danesi.
Drafting of the manuscript: Del Re, Danesi, Biasco.
Critical revision of the manuscript for important intellectual content:
Danesi, Jenster, van Schaik, Galli, Falcone.
Statistical analysis: Del Re, Biasco, Orlandini, Miccoli.
Obtaining funding: Danesi.
Administrative, technical, or material support: Biasco, Del Re.
Supervision: Danesi, Galli.
Other (specify): None.
Financial disclosures: Marzia Del Re certifies that all conflicts of interest,
including specific financial interests and relationships and affiliations
relevant to the subject matter or materials discussed in the manuscript
(eg, employment/ affiliation, grants or funding, consultancies, honoraria,
stock ownership or options, expert testimony, royalties, or patents filed,
received, or pending), are the following: None.
Funding/Support and role of the sponsor: The analysis described in this
article was funded by grants from the[3_TD$DIFF] Istituto Toscano Tumori (ITT;
Florence, Italy), Fondazione Cassa Risparmio di Lucca (Lucca, Italy), and
from the Italian Ministry of Instruction, University and Research (MIUR;
Rome, Italy) to Romano Danesi.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at
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