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Circula(ng microRNAs as diagnos(c marker in breast cancer pa(ents and their deregula(on under neoadjuvant chemotherapy Pierre Frères, Claire Josse, Stéphane Wenric, Nicolas Bovy, Meriem Boukerroucha, Ingrid
Struman, Vincent Bours, Guy Jerusalem
GIGA-­‐Research, Human Gene6cs, Université de Liège Breast cancer is the leading cause of death by cancer among women and there is an urgent need to improve its
diagnosis and prognosis. MicroRNAs (miRNAs) are non-coding RNAs that regulate gene expression and many
have been implicated in breast cancer. In this article, we focus on circulating miRNAs as biomarkers for breast
cancers and we describe the deregulation of their expression during neoadjuvant chemotherapy (NAC).
1. Circula(ng miRNAs signature as diagnos(c marker in breast cancer pa(ents 1.4
<0.01
<0.01
1.9
<0.01
<0.05
1.3
<0.001
<0.05
1.5
*** ***
0.005
0.000
Breast cancer patients
m
iR
-2
81
c
21
d
m
iR
-1
p
-3
t-7
24
iR
1.3
le
99
a-
-3
-1
<0.001
0.010
Healthy women
iR
<0.0001
m
<0.05
1.2
iR
-2
<0.05
m
m
0.09
****
0.015
**
**
-3
1.4
3a
1.2
0.1
3p
0.1
<0.010.0
9c
<0.05
22
** ****1.3
<0.05
<0.01
**
0.020
m
0.5
<0.01
m
miR-223
Receiver operating characteristic (ROC) curve derived from the
8miRNAs based diagnostic tool exhibited an area under the curves
miR-328
miR-34a
(AUC) of 0,95 in the validating cohort
1.0
miR-181c expression (2-ΔCt)
Fold-change
0.08
-1
miR-199a-3p
2.5
B <0.01
Figure0.071 : 81.3circulating miRNAs significantly dysregulated in
miR-374b
miR-423-3p
2. C
Effect of neoadjuvant chemotherapy (NAC) on miRNAs 1.2 patients
breast<0.05
cancer
<0.05
miR-425
FDR
<0.05
iR
A diagnostic model, based on 8 miRNAs (Fig. 1) measured in 71
miR-139-5p
breast cancer patients and 14 healthy women, was designed. The
miR-15b
validity of this classification model has then been confirmed on an
miR-197
independent cohort of 30 patients and 6 controls D&8$
P-value
1.5
miRNAs expression (2-ΔCt)
NA1$
miR-126
miRNAs expression in plasma
Upregulated miRNAs
iR
A
The expression of 188 plasma miRNAs was determined in 101
A with primary or metastatic breast cancer and 20
B healthy
patients
miRNAs
women. We found 116 miRNAs modified in both primary miR-106a
and
metastatic breast cancer patients compared to controls.
miR-122
<0.01
<0.01
1.8
<0.01
0.06
1.6
Circulating
miRNAs
expression was assessed in plasma and
2
!
tumor of 25 patients before and after the NAC. 25 plasma miRNAs
Downregulated miRNAs
are significantly
modified
after NAC (Fig. 2). Tumor suppressor
miR-34a
miRNAs
P-value
FDR
Fold-change
miR-34a is highly upregulated in plasma atAUC
the= 0.95
end of NAC (Fig.
miR-127-3p
<0.01
0.08
0.7
miR-122
sensibility
= 0.9
3A). This
miRNA
displays also an increased
expression
in the
1
miR-150
<0.05
0.06
0.5
specificity = 0.83
tumor<0.05
tissue
after
NAC in 7 patients with
pathological
partial
threshold
= 0.3
miR-29c
0.1
0.8
response
to0.06NAC0.6(Fig. 3B). This work demonstrates for the first
miR-324-3p
<0.05
A
B
time miR-34a
that
NAC
induces
of expression
miR-34ain tumor
in plasma
and
miR-34a
tissues
expression
in plasmaexpression
miR-329
<0.05
0.06
0.6
100 in the anti-tumor
80
0
****
*
tumor<0.05
tissue0.2that 0.7might be involved
effect of the
miR-376a
chemotherapy.
Studying
the kinetic80 of miR-34a expression during
60
miR-376c
<0.01
0.2
0.7
60
NAC <0.01
revealed
that its level is
especially increased after
miR-382
0.3
0.7
miR-451
40
anthracycline-based
chemotherapy
40 (Fig. 3C). Moreover, miR-34a
miR-409-3p
<0.01
0.2
0.7
-1
20
upregulation
after
anthracycline20 is correlated with troponin
miR-411
<0.05
0.1
0.8
20
25
30
35
40
upregulation
in NAC
treated patients,
suggesting a role of this
0
0
miR-451
<0.01
<0.05
0.5
Ct
miRNA in anthracycline-mediated cardiotoxicity (Fig. 3D).
!
NAC
NAC
Figure 2 : Circulating miRNAs significantly dysregulated after NAC
C
D
A
B
miR-34a expression (2-ΔCt)
miR-34a expression in tumor tissues
N
D
-8
A
1
Troponin T
0
1
0
0.1
1
10
100
1000
miR-34a expression (log10*2-ΔCt)
NAC
NAC
NAC
2
10
3M
-8
20
D
A
-8
D
A
N
30
-8
0
3
D
20
****
40
***
A
2
40
1
0
60
***
N
20
80
Correlation between upregulation of
plasma miR-34a and troponins T
miR-34a expression in plasma
50
A
1
40
*
N
60
100
miR-34a expression (fold-change)
miR-34a expression (2-ΔCt)
****
1
miR-34a expression (2-ΔCt)
miR-34a expression in plasma
80
N
N
D
A
-8
1
miR-34a expression (2-ΔCt)
Ct (NA1 - D-8)
miR-503
miR-34a expression (fold-change)
Figure
3 : miR-34a upregulation under
C
D NAC
Correlation between upregulation of
plasma miR-34a and troponins T
miR-34a expression in plasma
Conclusions****
50
***
***
3
1)  Accurate biomarkers for minimally invasive diagnosis of primary and metastatic
breast cancer was identified ;
2)  NAC induces expression of miR-34a in plasma and tumor tissue, which could be
involved in anti-tumor effect and cardiotoxicity of chemotherapy agents. Troponin T
40
30
20
2
1
10
0
0.1
3M
D
-8
A
2
N
N
A
1
0
1
10
100
1000
miR-34a expression (log10*2-ΔCt)
NAC
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