next-generation sequencing (ngs) of two independent...

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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


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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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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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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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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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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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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GOLDEN (N=269)109 TBT vs 160 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

######

##

### ### ###

###

### ##

###

###

Mean±SE, ###p<0.001, ##p<0.01 Mann-Withney test

next-generation sequencing (ngs) of two independent...

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