NASH Biomarker workshop 2018

Use of plasma PRO-C3, PRO-C5, and PRO-C6 for the diagnosis and follow-up of fibrosis stage in patients with nonalcoholic fatty liver disease (NAFLD)

Fernando Bril1, Srilaxmi Kalavalapalli1, Morten A. Karsdal2, Samuel J Daniels2, Diana J. Leeming2, Kenneth Cusi1

1Division of Endocrinology, Diabetes and Metabolism The University of Florida, Florida, USA. 2Biomarkers and Research, Nordic Bioscience, Denmark.

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Disclosures

• Diana J. Leeming and Morten Karsdal are full time employee and stock owner of Nordic Bioscience A/S.

• Samuel Daniels is an employee of Nordic Bioscience A/S.

• No further disclosures from co-authors

Non-Alcoholic Fatty Liver Disease (NAFLD)

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Non-Alcoholic Fatty Liver Disease (NAFLD)

➢ NAFLD is prevalent in approximately 25% of the global population

➢ Approximately 10–20% of patients with NAFLD have non-alcoholic steatohepatitis (NASH), an advanced form defined by presence of steatosis, hepatocellular ballooning, and lobular inflammation1

➢ NASH is a fibrogenic phenotype of NAFLD that may progress to cirrhosis and liver failure. A hallmark is excessive and disordered deposition of ECM in the liver including increased degree of cross-linking

➢ There is a need for non-invasive tests to detect fibrosis amongst individuals at high risk for developing NASH

Thannickal VJ et al. Am J Pathol 2014Dreamstime

Fibroblast

1. World Gastroenterology Organisation Global Guidelines. 2012. Available at:

http://www.worldgastroenterology.org/UserFiles/file/guidelines/nafld-nash-english-2012.pdf

Fibrosis is not just fibrosis!

Healthy Pathology

Portal triad

Central vein

Hepatocytes

Bridging fibrosis

Pericellular fibrosis

1,2,3,5,11 and 24

28, 6

4

7

17, 13, 23 and 25

8 & 10

18

26, 27

9, 12, 14,16,19,

20, 21, 22

Laminin

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FIBROBLASTS

EPITHELIAL/ENDOTHELIAL CELLS

BASEMENT MEMBRANE

INTERSTITIAL MATRIX

A COLLAGEN IS NOT JUST A COLLAGENThere are 28 collagens - some and good and some are bad…..

IV: Arresten, canstatin,

tumstatin, tetrastatin,

pentastatin, hexastatin

VI: Endotrophin

VIII: Vastatin

XV: Restin

XVIII: Endostatin

network

Karsdal MA et al, Adv Drug Del Rev, 2017

NORDIC BIOSCIENCE’S BLOOD-BASED NEO-EPITOPES ARE MEASURES OF DISEASE RELATED ECM TURNOVER, AND OTHER RELATED MARKERS

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Modified from Karsdal MA et al. Am J Physiol Gastrointest Liver Physiol 2015;308(10):G807-G830.

Introduction and objective

• Type III, V and VI collagen synthesis is upregulated in liver fibrosis

• PRO-C3, PRO-C5 and PRO-C6 are neo-epitope marker reflecting true type III, V or VI collagen formation, released by specific proteinase

• PRO-C3 is known to be diagnostic as well as related to progression of liver fibrosis in chronic liver disease.

Objective: To investigate the relation between changes in plasma ECM biomarkers in relation to change in fibrosis determined by liver biopsy. Furthermore, to relate such

markers to degree of liver fibrosis.

1. Nielsen MJ et al. Liver Int 2015;35:429–37

2. Nielsen MJ et al. PLoS One 2015;10:e0137302

C3M

Intact

Pro-C3

PIIINP

PIIINP-ab

PRO-C3-ab

C3-ab

Procollagen

Patients and Methods

• Cross-sectional study1,2

– 222 overweight and obese patients were included

– 60 patients had “No NAFLD” by magnetic resonance spectroscopy; Biopsy-confirmed NAFLD: 162)

• Double biopsy treatment study3,4

– 86 patients with NAFLD received therapy for 18 months with liver biopsy at baseline and repeated after therapy.

– Placebo: n=30, Vitamin E: n=26, Pioglitazone: n=7, Vitamin E + Pioglitazone: n=23.

• Biomarker assessments– Markers of type III, V and VI collagen formation (PRO-C3, -C5, and -C6) were

assessed in plasma were measured by competitive ELISA (Nordic Bioscience, Herlev, Denmark) in all patients at baseline and 18.

1. Bril F, et al. Hepatology. 2017;65:1132-1144. 2. Bril F, Diabetes Obes Metab. 2018. doi: 10.1111/dom.13285. 3. Cusi K, et. al. 2016;165:305-15. 4. Bril F. et. al.

78th Scientific Sessions American Diabetes Association (to be presented).

Patient Demographics

Cross-sectional study of NAFLDn=222

Treatment study of NAFLD, follow-upn=86

Treatment study:Change in PRO-C3 was related to change in liver fibrosis stage by histology

All patients were pooled regardless of therapy

PRO-C3 and PRO-C6 were the best at identifying liver fibrosis- Patients with biopsy proven NAFLD

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PRO-C3 was the most optimal marker for identification of advanced and moderate fibrosis

PRO-C3 in combination with HbA1c and insulin increased AUROC to 0.89 for advanced liver fibrosis

Conclusions

• The change in plasma PRO-C3 was correlated to change in liver histology over an 18 months treatment period

• Plasma PRO-C3 and PRO-C6 were able to identify patients with advanced and moderate liver fibrosis

• A combination of plasma PRO-C3 with insulin and HbA1c increased the AUC to 0.89 for identification of patients with advanced fibrosis

Thank you for your attention

ATS May 23, 2017 15

Acknowledgements

Nordic Bioscience

Morten A Karsdal

Samuel J Daniels

University of Florida

Fernando Bril

Kenneth Cusi

Srilaxmi Kalavalapalli

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Contact Diana J Leeming at djl@nordicbio.com