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Next-Generation Sequencing (NGS) of two independentcohorts identifies eleven circulating miRNAs for
diagnosis of NASH and fibrosis
Sven Francque1, Geneviève Cordonnier2, Frederic Texier2, Vlad Ratziu3,4, Stephen Harrison5, Pierre Bedossa6, Quentin Anstee7, Alice Roudot2, Sophie Megnien2,
Dean Hum2, Bart Staels8, Pierre Chaumat2, Remy Hanf2, Arun Sanyal9.
1Antwerp University, Antwerp, Belgium; 2Genfit, Loos, France; 3Hopital Pitié Salpétrière, Paris, France; 4Intitute of Cardiometabolism and Nutrition, Paris, France; 5Pinnacle Clinical Research, San Antonio, TX, United States; 6Department of Pathology, Hopital Beaujon, Paris,
France, 7Insitute of Cellular Medicine, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, United Kingdom; 8Institut Pasteur de Lille, Lille, France; 9Virginia Commonwealth University, Richmond, VA, United States.
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Disclosures
Consultant and/or lecturer for Roche, Gilead, Abbvie, Bayer, BMS, MSD, Janssen, Actelion, Astellas, Genfit, Inventiva, and Intercept
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Previous work
Circulating miRNA’s are potential candidates for non-invasive diagnosis of NASH with fibrosis
Two independent cohorts:GOLDEN-505 trial : 270 subjects screened with NAS=1-8, F=0-4.
OBESE (Antwerp cohort): 253 obese patients NAS=0-8, F=0-4.
mir34a, mir122a, mir200a in serum by RT-qPCR (TaqMan)
Relation between levels of circulating miRNA and histology
Performance of individual miRNA’s for diagnosis of:
To-Be-Treated (TBT) vs. Non-To-Be-Treated (NTBT): NASH + NAS ≥ 4 + F ≥ 2
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Patient Characteristics: GOLDEN vs. OBESE
GOLDEN OBESENTBT
Mean (SD)TBT
Mean (SD)
P valueStudent t test
NTBTMean (SD)
TBTMean (SD)
P valueStudent t test
Patient Number 161 109 202 50
% male 54% 54% 33% 54%
Age (years) 51 (12) 55 (11) 0.01 43 (13) 43 (13) NS
BMI (kg/m2) 31 (5) 31 (4) NS 39 (5) 44 (7) < 0.001
Waist Circ (cm) 104 (12) 106 (11) NS 119 (12) 132 (13) < 0.001
ALT 61 (41) 76 (47) 0.008 35 (20) 66 (40) < 0.001
AST 37 (19) 54 (31) < 0.001 23 (9) 45 (29) < 0.001
Alkaline Phosphatase 82 (40) 76 (25) NS 83 (23) 85 (23) NS
GGT 96 (212) 87 (106) NS 43 (27) 65 (45) < 0.001
CK18-M30 534 (411) 843 (676) < 0.001 179 (116) 486 (508) < 0.001
Fasting Glucose 5.6 (1.3) 6.2 (1.6) 0.001 4.9 (0.9) 5.9 (2.8) < 0.001
Fasting Insulin 155 (122) 204 (188) 0.02 126 (72) 227 (200) < 0.001
HbA1c 5.8 (0.7) 6.3 (1.0) < 0.001 5.6 (0.6) 6.1(1.0) < 0.001
Steatosis (0-3) 1.9 (0.9) 2.4 (0.6) < 0.001 1.2 (0.9) 2.4 (0.7) < 0.001
Lob. Inflam. (0-3) 1.1 (0.5) 1.7 (0.6) < 0.001 0.8 (0.8) 1.9 (0.8) < 0.001
Ballooning (0-2) 1.1 (0.6) 1.6 (0.5) < 0.001 0.8 (0.8) 1.4 (0.5) < 0.001
NAS (0-8) 4.0 (1.5) 5.7 (1.0) < 0.001 2.8 (2.2) 5.7 (1.3) < 0.001
Fibrosis (0-4) 0.9 (0.7) 2.5 (0.5) < 0.001 0.4 (0.6) 2.4 (0.5) < 0.001
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miRNA levels in TBT vs. NTBT patients
GOLDEN-505 OBESE
1
2
3
4
***
log1
0 co
pies
.µL-1
miR
-34a
-5p
0
1
2
3
***
log1
0 co
pies
.µL-1
miR
-200
a-3p
2
3
4
5
6*
log1
0 co
pies
.µL-1
miR
-122
a-5p
NTBT TBT
1
2
3
4
***
log1
0 cop
ies.µL
-1 m
iR-3
4a-5
p
2
3
4
5
6
***
log1
0 cop
ies.µL
-1 m
iR-1
22a-
5p
0
1
2
3
log1
0 cop
ies.µL
-1 m
iR-2
00a-
3p
NTBT TBT
mir34a
mir122a
mir200a
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miRNA ROC Curves: TBT vs. NTBT
GOLDEN-505 OBESE
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Aims
Unbiased identification of miRNA’s differentially expressed in serum samples from patients with NASH and fibrosis (TBT) vs. NTBT.
Methods
Circulating levels of 2083 miRNA species were simultaneously measured and discriminating miRNA’swere then confirmed by a classical RT-q-PCR approach.
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HTG-EdgeSeq-NGS
GOLDEN-Diag (N=271)110 TBT vs 161 NTBT
OBESE (N=249)50 TBT vs 199 NTBT
Ove
r ex
pre
ssed
Ove
r ex
pre
ssed
Un
der
exp
ress
ed
Un
der
exp
ress
ed
Volcano-plots obtained in GOLDEN-Diag and OBESE comparing fold change and p value of each individual miRNA in TBT vs. NTBT patients
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HTG-EdgeSeq-NGS
There were more over-expressed than under-expressed miRNA’s whenselection was based on p<0.01:
34 over-expressed vs. 14 under-expressed in GOLDEN-505 cohort17 over-expressed vs. 5 in OBESE cohort
When selection of over-expressed miRNAs was based both on statisticalsignificance (p<0.01) and fold change (>1.3) in TBT vs. NTBT
21 were selected in GOLDEN-50514 were selected in OBESE
After removing miRNAs with low expression levels in GOLDEN (<100 readsin both TBT and NTBT), cross-validation between the two cohorts
-> Commonly over-expressed miRNA in TBT vs NTBT:
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HTG-EdgeSeq-NGS
GOLDEN (N=269)109 TBT vs 160 NTBT
OBESE (N=248)50 TBT vs 198 NTBT
Fold Change p-value Fold Change p-value
miR-34a-5p 1.92 1.3E-10 2.18 5.5E-11
miR-A 1.76 8.1E-09 1.96 1.2E-11
miR-B 1.55 4.9E-07 1.81 1.7E-04
miR-C 1.38 4.2E-06 1.45 7.0E-06
miR-D 1.37 1.8E-05 1.52 5.6E-05
miR-E 1.33 2.4E-05 1.31 6.6E-03
miR-F 1.37 2.9E-05 1.40 7.2E-04
miR-122-5p 1.50 2.0E-04 2.40 1.5E-08
miR-G 1.38 8.6E-04 1.34 4.0E-02
miR-H 1.37 1.2E-03 1.62 2.7E-04
miR-I 1.44 1.7E-03 1.48 7.5E-03
Newly discovered miRNAs are coded : patent filling ongoing
Commonly over-expressed miRNAs (TBT vs. NTBT; fold change >1.3 and p<0.01) in GOLDEN and OBESE cohorts
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Confirmation of mir34a and mir122 by RT-qPCR
GOLDEN-Diag
OBESE
###
###
### ###
######
Mean±SE, ***p<0.001, Mann-Whitney test
Discriminating potency of mir34a in GOLDEN-Diag and OBESE onTBT vs. NTBT, NAS ≥ 4 vs. NAS < 4 and F ≥ 2 vs. F < 2
Mean±SE, ###p<0.001, ##<p<0.01, #p<0.05, Mann-Withney test
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Confirmation of mir34a and mir122 by RT-qPCR
GOLDEN-Diag
OBESE
***
*
*****
*
*****
*****
mir34a serum level increases with NAS and Fibrosis stageMean±SE, ***p<0.001, **<p<0.01, *p<0.05, Dunn’s test
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Confirmation of mir34a and mir122 by RT-qPCR
GOLDEN-Diag
OBESE
####
#
### ######
Discriminating potency of mir122 in GOLDEN-Diag and OBESEon TBT vs. NTBT, NAS ≥ 4 vs. NAS < 4 and F ≥ 2 vs. F < 2
Mean±SE, ###p<0.001, ##<p<0.01, #p<0.05, Mann-Withney test
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Confirmation of mir34a and mir122 by RT-qPCR
GOLDEN-Diag
OBESE
*
***
**
****
mir122 serum level increases with NAS and Fibrosis stageMean±SE, ***p<0.001, **<p<0.01, *p<0.05, Dunn’s test
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Confirmation of newly identified miRNA’s by RT-qPCR
GOLDEN-Diag
OBESE
###
###
**
*
***
***
*****
***
*
Discriminating potency of mir-A and correlations with NAS and Fibrosis stageMean±SE, ###p<0.001, Mann-Withney test; ***p<0.001, **<p<0.01, *p<0.05, Dunn’s test
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Confirmation of newly identified miRNA’s by RT-qPCR
GOLDEN-Diag
OBESE
###
###
***
*****
Discriminating potency of mir-C and correlations with NAS and Fibrosis stageMean±SE, ###p<0.001, Mann-Withney test ***p<0.001, **<p<0.01, *p<0.05, Dunn’s test
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Confirmation of newly identified miRNA’s by RT-qPCR
GOLDEN-Diag
OBESE
###
###
***
**
*****
**
Discriminating potency of mir-F and correlations with NAS and Fibrosis stage.Mean±SE, ###p<0.001, Mann-Withney test; ***p<0.001, **<p<0.01, *p<0.05, Dunn test
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Confirmation of newly identified miRNA’s by RT-qPCR
GOLDEN (N=269)109 TBT vs 160 NTBT
OBESE (N=248)50 TBT vs 198 NTBT
Fold Change p-value AUROC Fold Change p-value AUROC
miR-34a-5p 1.99 <0.001 0.74 1.54 <0.001 0.74
miR-A 1.80 <0.001 0.68 2.85 <0.001 0.74
miR-C 1.52 <0.001 0.68 1.41 <0.001 0.65
miR-F 1.65 <0.001 0.65 1.88 <0.001 0.68
miR-122-5p 1.15 <0.01 0.59 2.39 <0.001 0.77
miR-G 1.65 <0.01 0.61 1.18 <0.01 0.62
miR-H 1.25 <0.01 0.63 1.12 <0.01 0.62
Newly discovered miRNAs are coded : patent filling on going
Fold change and AUROC of individual miRNA’s dosed by RT-qPCR
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Conclusion
By analyzing serum samples of more than 500 NAFLD patients from twoindependent cohorts by NGS technology , this large and exhaustive study has allowed a non-biased selection of the most discriminating circulating miRNA’sassociated with NASH and liver fibrosis.
Fom a total of 2083 miRNA’s reffered in mir-Base, 11 miRNA’s have been shown to be significantly over expressed in TBT vs. NTBT in two indepedent cohorts of patients.
In addition to miR34a and mir122, 9 new miRNA’s were significantly associatedwith TBT condition, NAS≥4 and F≥2.
RT-qPCR experiments confirms that serum levels of selected miRNA’s graduallyincreased with NAS and fibrosis score.
These miRNA’s hold promise for developping new in vitro diagnostic tests to non-invasively screen NAFLD patients and identify NASH patients to be treated.
Reproducing performance in 2 cohorts with different selection methodology and disease distribution reinforces value as tools in diagnostic appraoch.
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Contributors
Thank you for your attention
GENFITLoos, France
Genevieve Cordonnier
John Brozek
Fouad Ben Sudrik
Dean Hum
Sophie Megnien
Alice Roudot
Raphael Darteil
Rémy Hanf
UZAPeter Michielsen
Luisa Vonghia
Luc Van Gaal
An Verrijken
Ilse Mertens
Vlad RatziuInstitute of Cardiometabolism and Nutrition
Hôpital Pitie Salpetrière, Paris, France
Stephen HarrisonUniversity of Oxford, UK
Quentin AnsteeFaculty of Medical sciences
Newcastle University, Newcastle upon Tyne, UK
Pierre BedossaHôpital Beaujon Paris, France
Bart StaelsINSER U1011, EGID, Université Lille-2, Lille, France
Arun SanyalVirginia Commonwealth University, Richmond, VA, USA
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RT-qPCR confirmation of new miRNA’s
GOLDEN-Diag
OBESE
mir
A
######
##
### ### ###
###
### ##
###
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Mean±SE, ###p<0.001, ##p<0.01 Mann-Withney test