W​HITE​ P​APER
Validation of an Off-the-Shelf, Diet-Induced NASH Mouse Model using Digital Whole
Slide Scanning of Liver Tissue and Artificial Intelligence-Enabled, Quantitative
Histopathological Analysis
Daniel Schroen, PhD, Commercial Lead, R&D, Reveal Biosciences (corresponding author, dschroen@revealbio.com)
Susan Kelly, HTL(ASCP)QIHC, Histology Group Lead, Reveal Biosciences
Manindra Singh, PhD, Scientist I, Explora BioLabs
Brandy Wilkinson, PhD, Director Breeding and Research, Explora BioLabs
Christopher Coley, Marketing Development Manager, Digital Pathology, Epredia
Sara Becker Catania, PhD, Associate Director, Product Development, Reveal Biosciences

S​UMMARY
Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are common chronic liver disorders resulting in lipid
accumulation in the liver, inflammation, hepatocyte damage, and fibrotic scarring, all of which impact organ function. With the lack ofapproved therapies for NASH patients, and considerable inter-pathologist variability in the scoring and semi-quantitative assessment of
the pathology used in their diagnoses, much focus and effort is being placed in the development of rodent models, the assessment of
individual disease features in these models, and understanding the effect of therapeutics on the disease process. The study describedin this paper is the result of a unique collaboration between several companies with combined goals of providing the NASH researchcommunity with a time-efficient, flexible, off-the-shelf diet-induced mouse NASH B6 Model (Taconic Biosciences, Inc.) that can beutilized in directed research studies for therapeutic drug development (Explora BioLabs). The results were digitally scanned using a
3DHISTECH PANNORAMIC™ series scanner (Epredia) and validated with imageDx NASH, a digital assay platform featuring machineand deep learning models to quantify steatosis, inflammation, hepatocellular ballooning, and fibrosis (Reveal Biosciences, Inc).
AI-generated quantitative results were compared with qualitative NAS scores provided by a board-certified veterinary pathologist toevaluate grade and fibrosis stage. The AI-based digital analysis results of the treated and untreated diet-induced NASH B6 model
correlated with the CRN scores recorded by the experienced veterinary pathologist. Importantly, the quantitative data also
demonstrated measurement of more granular pathologic changes. These subtle - but likely significant - pathologic changes are notcurrently well-captured by the standard scoring system, and underscore the need for and the utility for sensitive and reproducible
quantitative analysis.

I​NTRODUCTION
NAFLD/NASH
Non-alcoholic fatty liver disease (NAFLD) is one of themost common causes of chronic liver disease andresults in an accumulation of fat in the liver, often withfew or no symptoms. NAFLD can progress to a more
pathologically significant form of NAFLD known as
non-alcoholic steatohepatitis (NASH), characterized byhepatitis (inflammation) and hepatocellular ballooning(cell injury), which can lead to excessive fibrosis andscarring. NASH has the potential to progress to more
extensive
pathologic
featuresincludingportalhypertension, cirrhosis and hepatocellular carcinoma,
resulting in impaired liver function that often requires
liver transplantation as treatment. The incidence of
NAFLD/NASH has increased in the last ten years and is
growing, at least partially due to increasing incidence ofrisk factors such as type 2 diabetes, insulin resistance,

metabolic syndrome, and patients with high bloodpressure, cholesterol, lipids and triglycerides.

Mouse models in NASH research
At present, the US Food and Drug Administration (FDA)
has yet to approve any therapies for the treatment of
NASH. However, with the increased incidence of NAFLD
and NASH, the biopharmaceutical industry has
expressed active interest in developing drugs andtreatments for NASH, with initial studies requiring theuse of animal models. Although multiple mouse modelsof NAFLD/NASH exist, they have been limited by thelack of well-validated animal models that accurately
mimic the human disease’s multifactorial features. The
ideal experimental mouse model would closely replicate
human macrovesicular steatosis, hepatocyte ballooning,and fibrosis, and respond in a fashion similar to howpatients would react to therapeutic interventions. A
number of drug candidates, spread throughout phase
I-III clinical trials, include inhibitors, receptor agonists,
and antibodies, used singly or in combination. Theirtargets include insulin resistance, gluconeogenesis,
lipogenesis and lipid transport, all targeting steatosis andreduction of lipid accumulation. At more severe stages of
disease, therapeutics are directed towards apoptosis,
oxidative stress and inflammation, all hallmark featuresof steatohepatitis. At the most progressive stage of
disease, therapeutics address pathways that result inexcessive deposition of extracellular matrix (ECM)
components, with the aim to thwart or reverse cirrhosis.
Animal models that successfully recapitulate thesestages of the human disease continuum provide the best
opportunity for therapeutic development.
As a readout for therapeutic treatment, NASH/NAFLD
mouse models are either induced by diet (obesogenic,
nutrient deficient, or chemically induced), and/or are
genetically predisposed to develop the phenotypiccharacteristics of NASH. However, many of thesemodels do not successfully establish all features of thehuman disease. Diet-induced models such as the
Taconic Diet Induced NASH B6 develop the
NAFLD/NASH phenotype following extended ingestionof a high fat, high sugar, high cholesterol diet. One
limitation of current diet-induced models is the amount oftime it takes to evolve features of NAFLD/NASH. With
the recent availability of off-the-shelf NASH B6 mice
(Taconic Biosciences) already on a modified Amylin
Liver NASH (NASH) diet, researchers can reduce thetime to development of steatosis, inflammation, andfibrosis with a consistent and cost-effective approach. In
this study, NASH B6 mice are treated with two different
therapeutic candidates. Elafibranor, a duel peroxisome
proliferator-activated receptor (PPAR) agonist, targets
PPARalpha and PPARdelta, primarily affecting
hepatocyte ballooning and inflammation (Ratziu et al.
2017). Obeticholic acid (OCA), a semi-synthetic bile acidanalog, functions as a farnesoid X receptor agonist,
thereby decreasing the amount of bile in the liver andimproving steatosis, inflammation, and fibrosis (Makri etal. 2016).

Analysis of NASH features
The increasing incidence of NASH, and its associatedpotential for more serious liver disease, has compelled
development of new methods to detect NASH earlier, todifferentiate NAFLD from NASH, and to ensure reliable,consistent measurements of the disease process in

preclinical animal studies. In addition to the challenges
associated with generating validated mouse models, one
must
also
ensureconsistentandunbiasedmeasurements of pathologic features. Along with
monitoring body weight and blood analytes, liver biopsy
and associated histologic analysis remains the gold
standardfordiagnosing
NASH/NAFLD.
Semi-quantitative reporting of steatosis, inflammation,
ballooning, and fibrosis is typically done using the NASH
CRN grading and staging system (Kleiner et al. 2005).
Although biopsy and subsequent histologic staining ofsections is quite routine, the analysis and stage
designation for each NAFLD/NASH feature can bemarkedly subjective. Inter-pathologist concordancestudies indicate low Kappa Scores for different histologic
features, with published data ranging from 0.45-0.84(Kleiner 2005), 0.41-0.61 (Bedossa 2014), and 0.46-0.77(Kleiner 2019). The NAFLD activity score (NAS), widelyused to assess pathology in biopsies, is an 8-point scalemeasuring steatosis, ballooning, and inflammation.
However, subjectivity, its semi quantitative nature, andhigh interobserver variability limit the NAFLD activity
score, which has not been shown to have prognostic
value. To address subjectivity in liver pathology staging,
Reveal Biosciences has developed a novel digital assay
providing quantitative analysis for each of the pathologic
features of NAFLD/NASH. This approach leverages bothmachine learning (ML) and deep learning to provide amore quantitative and less-subjective approach tomeasure and stage liver pathology.

ImageDx™ NASH Analysis
Reveal Biosciences launched the imageDx™ NASH
digital assay platform in 2019, a project developed inclose collaboration with a team of leading US liver
pathologists to support both the pre-clinical and researchsector, as well as to provide support for NASH clinicalpathologists. As a cloud-based decision support tool for
quantitative NASH data, imageDx greatly enhances
project accuracy, reproducibility and scale. This platform
provides image hosting with broad geographical access,facilitating collaboration, image sharing, annotation andanalysis. Based on precise, consistent machine anddeep learning, imageDx yields accurate quantification
across samples, with AI-based NASH models trained
from thousands of image patches. In the imageDx
workflow, tissue samples are processed at Reveal Bio or
any quality histopathology lab, after which whole slide
images are generated and uploaded to the imageDx
cloud for analysis. Custom analysis and continuous,quantitative results are then produced for review. Built-in

quality controls automatically detect and exclude tissue
artifacts, folds, tears, out-of-focus regions, and slidedebris that may otherwise impact data quality.
While the most applicable mouse NASH models
replicate pathology seen in human NASH liver, somedifferences are inherent to mouse tissues. Therefore, theimageDx™ NASH digital assay was developed to
specifically address species differences. Individual AI
scoring algorithms have been developed and validated
specifically for rodent and human liver to generate
accurate analysis. For staging of rodent NASH liver

samples, the imageDx™ NASH digital assay offers anon-subjective, validated alternative or addition topathologist review of stained liver samples. The analysis
uses hematoxylin and eosin (H&E), Masson’s trichrome(MTC) or Picrosirius red (PSR)-stained slides for the
analysis of steatosis, ballooning, inflammation, fibrosis
and the presence of Mallory bodies. In this study, thequantitative results from the imageDx™ NASH analysis
were run in parallel and compared to results from aboard-certified veterinary pathologist.

M​ATERIALS​ ​AND​ M​ETHODS
Starting at 6 weeks of age, thirty (30) C57BL/6NTac male mice (Taconic Biosciences, Inc., Rensselaer, NY, USA) wereconditioned for 28 weeks (at the vendor) on high-fat, modified Amylin liver NASH rodent diet D09100310i (Research
Diets, Inc., New Brunswick, NJ USA), containing 40 kcal% Fat (Palm Oil), 20 kcal% Fructose and 2% Cholesterol.
Following arrival at the study site (Explora BioLabs) during week 28 of diet conditioning, animals were acclimatized for 2
more weeks until week 30 at which time a group of animals was sacrificed for baseline data and another group wasselected for an 8-week treatment beginning in week 31 with the animals sacrificed at week 39. Animals on the NASH dietwere randomized and either left on the NASH diet to serve as a vehicle control group, or treated daily for 8 Weeks, PO,with 10mg/kg Elafibranor (Genfit, Loos, France) or 30mg/kg Obeticholic Acid (Intercept Pharma, New York, NY USA). Five
(5) age-matched, control mice were fed standard diet NIH-31M chow. Liver morphology, serum chemistry (liver panels)
were examined and analyzed (Explora BioLabs, San Diego, CA, USA). Biopsied or whole mouse livers were fixed,
processed, embedded, sectioned, mounted on slides and stained with Hematoxylin and Eosin (H&E), Masson’s trichrome(MTC), and Picrosirius red (PSR) using optimized and validated staining protocols (Reveal Biosciences, San Diego, CA,
USA). Whole slide images were scanned and imaged under brightfield at 40x magnification with a 3DHISTECHPANNORAMIC™ FLASH 250 (Epredia, Kalamazoo Michigan USA). The study design is summarized below.

Timepoint

Number of Animals for
30, 39 Week Timepoints

StainsStandard Diet (NIH-31M)

30, 39 Weeks

5, 5

H&E, MTC/PSR

NASH Diet30, 39 Weeks

10, 5

H&E, MTC/PSR

NASH Diet + Elafibranor

39 Weeks

5

H&E, MTC/PSR

NASH Diet + Obeticholic Acid

39 Weeks

5

H&E, MTC/PSR

Treatment

Highly quantitative data were generated using imageDx™ NASH (Reveal Biosciences), an automated, integrated
workflow that analyzes and manages whole slide images. Figure 1 summarizes the process that Reveal Biosciences
follows to obtain analysis data. Each whole slide image is first assessed for quality using a precise focal measurement
followed by an accuracy check. All tissue and staining artifacts are digitally excluded from the reported quantification. The
analysis process includes automated identification of tissue, followed by segmentation of regions of interest and thenclassification of stained positive and negative cells and structures for measurement of expression. The identified regions
were then quantified for precise positivity.

Figure 1. ​Reveal Bioscience Digital Assay Workflow

Images were quantified for steatosis (% by area, % macrovesicular and microvesicular, average vesicle size),inflammation (immune cell density, immune cell count), hepatocellular ballooning (ballooning hepatocyte density),presence or absence of Mallory bodies, fibrosis (% by area), as summarized below. A summary of numerical data fromthe quantitative imageDx™ NASH analysis is presented in the appendix.

Steatosis
Analysis

The amount of total lipid accumulation, subcategorized into macro or micro
vesicular, amount of lipid/hepatocyte, and the mean vesicle size within theentire section area analyzed of the H&E stained section.

Ballooning
Hepatocyte Density

The density of ballooning hepatocytes within the entire section area
analyzed of the H&E stained section.

Immune Cell
Density

The total number of inflammatory cells and inflammatory cell density within
the entire section area analyzed of the H&E stained section.

Fibrosis
Percentage (%)

The amount of total fibrosis within the entire section area analyzed of the
MTC or PSR stained section.

Mallory Bodies

The presence or absence of Mallory Bodies within the entire section area
analyzed of the H&E section.

Tissue Area
Analyzed (mm^2)

The total tissue area used for analysis in the sample. This metric excludes
any region classified as artifact, out of focus or otherwise purposely
excluded from the analysis and is used for each feature analysis.

For microscopic evaluation of liver sections, a triple board-certified veterinary pathologist with experience in laboratory
animals and toxicologic pathology evaluated the H&E, PSR, and MTC-stained slides. The H&E-stained sections wereused to assess steatosis (fatty change), inflammation, hepatocellular ballooning, bile duct hyperplasia, and extramedullary
hematopoiesis (EMH). The PSR and MTC-stained sections were evaluated for fibrosis, indicated by various shades ofblue with darker blue indicating mature collagen and pale blue indicating newer or immature collagen. Samples submitted
for evaluation included 2 sets of slides of liver biopsies (2 images each for livers stained with H&E and MTC or PSR
stains).

R​ESULTS
Diet induced- NASH B6 mice exhibit increased body and liver weightAs shown in Figure 2, the diet-induced NASH mice fed the NASH diet exhibited significantly higher body weight compared
to animals fed the standard control diet (2A). From a morphological standpoint, livers from the NASH diet animals
appeared larger in size and lighter in color (2B), characteristic of NASH. Liver weights, normalized as a percentage ofbody weight, were well above those of control animals (2C).

A.

​Body Weight (28-30 Weeks)

B.

C.

​Liver Weights (Week 30)
p<0.0001

Figure 2. ​Characterization of NASH diet conditioned animals at 30-week timepoint shows (A) decrease in weight of NASH diet animals
at arrival at study site followed by an increase in body weight through the 2-week acclimatization period. At the 30-week timepoint,
NASH conditioned animals show significantly higher body weights compared to control diet animals (p<0.0001, two-way ANOVA), (B)liver morphology with increased organ size of the NASH diet animals and (C) significant increase in liver weight compared to control
diet animals (p<0.0001, unpaired t-test).

Steatosis Analysis
For steatosis quantitation, lipid regions (white) within the H&E-stained slides were identified and quantitated as a percentof the total image analysis area. The algorithm first identified tissue areas on whole slide images followed by a more
localized identification of positive lipid regions. Lipid regions are visualized by the blue mask overlaid onto the tissue(Figure 3A). The amount of lipid or steatosis was measured and expressed as a percentage of the total image analysis
area. The amount of lipid was subcategorized into macrovesicular and microvesicular steatosis, as determined by lipid
droplet size with a cutoff of 65 ​µ​m2​ (Figure 3B). The percentage of steatosis area and macrovesicular steatosis in the
NASH diet animal sections was significantly increased compared to that measured in the control diet animals at 30-weeks(Figure 3C). The full data set includes percent lipid area, total lipid area (mm​2​), percent macrovesicular, percent
microvesicular, average vesicle size (​µ​m2​​ ), lipid/hepatocyte, and tissue area analyzed (Appendix Tables 1-2). The
precision of the steatosis quantification was measured by evaluating the mean lipid area and the mean vesicle size intriplicate with data at each experimental timepoint. Across control diet and NASH diet fed animals, the mean liver lipid
area varied from 0.03-0.09% and 0.00-0.08% and the mean droplet size varied from 0.01-0.05% and 0.02-0.06%

respectively, indicating that the steatosis algorithms perform in a highly precise manner for the determination of lipid
amount and mean droplet size with control and treated tissue section images.

A.

B.

C.
​

Steatosis

Macrovesicular

Microvesicular

D.
Steatosis Pathology Score
0 < 5%
1 5% to 33%
2 > 33% to 67%
3 > 67%This Study at Weeks 30, 39

Score 3 in all NASH diet sections; hepatocytes with both microvesicular andmacrovesicular fatty vacuoles in the cytoplasm. 0 in all control NIH31M diet sections at
30-weeks. At 39-weeks, Score 3 in NASH vehicle, Elafibranor, and OCA treatmentgroups.

Figure 3. ​Steatosis analysis of NASH diet conditioned animals at 30-weeks (A) showing liver stained with H&E (A, left) andcorresponding mask, the result of cell-by-cell segmentation (A, right) over the same section of tissue identifying cells positive for lipid
accumulation (blue) used for quantification, (B) Hepatocytes with fatty change (steatosis); examples of macrovesicular (blue arrows)
and microvesicular (green arrows) lipid accumulation within hepatocytes, all NASH diet sections showed similar pathology. (C)Quantitation of total percentage of steatosis shows markedly elevated amounts in NASH diet animals compared to control diet withsignificant differences in the amounts of macro- and micro-vesicular steatosis (p<0.0001, unpaired t-test, all steatosis), (D) Pathologist
description of steatosis and score summary for control, NASH, NASH with Elafibranor, and NASH with OCA treatment groups.

The results of the lipid quantitation show that the mean lipid per hepatocyte increased 80-fold from 3.31 ​µ​m2​ to 266.9 ​µ​m2​
in the animals fed the NASH versus the standard NIH-31M diet for 30 weeks (Figure 4A). The mean vesicle size
increased more than 2-fold from 34.2 ​µ​m2​ to 76.0 ​µ​m2​ in the animals fed the NASH versus the standard NIH-31M diet(Appendix Tables 1,2). The animals fed the NASH diet mainly showed macrovesicular accumulation of lipid within
hepatocytes versus the control diet group which primarily had very small microvesicular lipid inclusions within
hepatocytes. At the 39-week timepoint (8 week treatment), the mean lipid per hepatocyte in the vehicle control NASH dietgroup was 212.16 ​µ​m2​ ​compared to 45.47 ​µ​m2​ and 78.53 ​µ​m2​ in the Elafibranor and the OCA treatment groups
respectively indicating a significant reduction in the amount of lipid retained within hepatocytes with the liver samples(Figure 4B).

A.

B.
Mean Lipid/Hepatocyte (Week 30)

Mean Lipid/Hepatocyte (Week 39)

Figure 4. ​Mean lipid per hepatocyte (um​2​) averaged over entire whole slide image for all animals within control (n=5) or NASH dietgroups (n=10) at 30-weeks (A) or post-treatment 39 weeks (B). (A) Mean lipid/hepatocyte increases significantly (80-fold) with NASH
diet compared to standard NIH-31M diet (p<0.001, unpaired t-test). (B) Mean lipid/hepatocyte in NASH Elafibranor and OCA-treatedgroups show significant decrease in the amount of lipid per hepatocyte compared to the NASH diet vehicle control group at 39-weeks(p<0.0001, unpaired t-test).

The pathologist NAS score for steatosis is based on the number of hepatocytes with either macrovesicular and/or
microvesicular hepatocellular fatty vacuoles. Steatosis (hepatocellular fatty change) was a notable feature of the livers inthis study. Fatty vacuoles were seen as clear intracytoplasmic vacuoles with clear, intact edges. Microvesicular fatty
vacuoles were seen as approximately 1μm or smaller single or multiple vacuoles; macrovesicular vacuoles were seen as
3 μm or larger and often single but sometimes multiple within the same cell. Many of the fatty vacuoles seen weremicrovesicular. At the 30-week timepoint, the NIH-31M control diet fed animals scored a 0 and the NASH diet fed animals
scored a 3, indicating either less than 5% steatosis or more than 67% steatosis respectively (Figure 3D). Within each ofthese groups, the pathologist-derived score was consistent for all animals. At this timepoint, the average of the AI-derived
lipid area expressed as a percentage of tissue area analyzed for control diet animals was 0.31% and 15.1% for NASH dietanimals showing a similar trend to the pathologist-reported lipid percentages but a gross correlation with actual
percentage values per group (ex. the NASH diet group with AI-reported value of 15.1% vs. pathologist-reported value ofgreater than 67%). At the 39-week timepoint, pathologist-reported steatosis was more than 67% for the NASH vehicle and
NASH diet groups treated with Elafibranor and OCA (Score 3) while the AI-derived lipid percentages were 13.2% for
NASH vehicle, 3.1% for NASH with Elafibranor and 6.6% for NASH with OCA treatment. The AI-measured difference inlipid percentages for the vehicle and the Elafibranor and OCA-treated groups was significant (p<0.01, unpaired t-test) andin comparison to the pathologist-reported scores, shows that the AI-driven approach provides a more sensitive
measurement of lipid change in untreated and treated tissue sections.

Hepatocyte Ballooning Analysis
Ballooning hepatocytes were identified and quantitated as the number of ballooning cells within the total image analysis
area of H&E-stained liver sections. The algorithm first identified tissue areas on whole slide images followed by positive
ballooned cells based on the cell diameter and presence of disrupted cytoskeletal structure, an example is shown (bluebox, Figure 5A). The full data set includes the density of ballooning hepatocytes (cells/cm​2​), and the tissue area analyzed(mm​2​) (Appendix Tables 3&4).

A.

B.
​H&E NASH Diet

Ballooning Cell NASH Diet

Ballooning Hepatocytes (Week 30)

C.
Ballooning Hepatocytes (Week 39)

D.
Ballooning Pathology Score
0 None
1 Few
2 ManyThis Study at Weeks 30, 39
At 30-weeks, ballooning in NASH diet sections was 0 (2/10), 1 (2/10), and 2 (6/10) animals.
Control group standard NIH-31M diet had no ballooning (0) in all sections. At 39-weeks, noballooning (0) was found in the standard NIH-31M control diet, NASH Elafibranor, and the
NASH OCA-treated group had 1 animal with Score 1. The NASH vehicle group had a rangeof Score 0 (2/5) and 1 (3/5).

Figure 5. ​Hepatocyte ballooning analysis of NASH diet conditioned animals at 30-weeks (A) showing liver stained with H&E (A, left)
and corresponding blue box, the result of cell-by-cell segmentation (A, right) over the same section of tissue identifying cells positive forballooning across the entire whole slide image used for quantification. (B) Quantitation of the frequency of ballooning hepatocytes
shows markedly elevated numbers in NASH diet animals compared to control diet (p<0.0001, unpaired t-test) at 30-weeks (C) At
week-39, frequency of ballooning hepatocytes is increased in NASH diet vehicle group compared to control diet (p<0.001, unpaired
t-test), frequency of ballooning hepatocytes is decreased in the NASH Elafibranor and OCA-treated groups compared to NASH dietvehicle group following treatment (p<0.001 and p<0.05 respectively, unpaired t-test) (D) Pathologist description of ballooning and scoresummary for control, NASH diet, NASH Elafibranor, and OCA-treated groups.

The results of the ballooning hepatocyte analysis show that in both the 30- and the 39-week studies, there are negligible
numbers of ballooning hepatocytes within the standard diet (NIH-31M) control groups at both timepoints (Figure 5B and
5C). Within the NASH diet groups, the 30-week group had a mean ballooning hepatocyte density of 18.82 cells/cm​2 while
the NASH diet vehicle control group at the 39-week timepoint had a mean ballooning hepatocyte density of 57.32
cells/cm​2 which shows an increase in numbers of ballooning hepatocytes during the 8-week treatment period during whichanimals are fed the NASH diet and hepatocytes are accumulating lipid which is causing cytoskeletal injury and theresultant ballooning phenotype. The 8-week treatment of NASH diet animals with both Elafibranor and OCA significantly
reduced the frequency of ballooning cells to 8.15 cells/cm​2 and 18.86 cells/cm​2 compared to the NASH vehicle diet group(57.32 cells/cm​2​).
The pathologist description of hepatocyte pathology was reported as a range of changes, from marked hydropic
degeneration with lacy indistinct intracytoplasmic vacuoles, to greatly expanded cells with degenerate or no nuclei, to lossof cells with only faint (ghost) outlines of cells remaining (Figure 5D). Cells with loss of nuclei or very pale or condensed
nuclei and/or obvious cytoplasmic enlargement with notable cell debris were indicative of necrosis. Within the 30-week
timepoint, the pathologist-assigned ballooning score was 0 for all animals on the standard NIH-31M diet and varied from 0(2 animals), 1 (2 animals) to 2 (6 animals) for those on the NASH diet (Figure 5B). In comparison, AI-derived ballooning
hepatocyte densities were 0 for animals on the NIH-31M diet and averaged 18.82 cells/cm​2 ​for those animals fed the
NASH diet. Within the NASH diet group, the ballooning hepatocyte density ranged from 11.84 to 37.29 cells/cm2 showing
the amount of individual animal variation that can be measured using the AI-derived method versus a numerical scorewith poor definition of the difference between scores. Within the Elafibranor and OCA treatment groups at the 39-week
timepoint, the pathologist ballooning hepatocyte scores were mostly 0 with a single animal scored 1. The AI-derived
quantitation of hepatocyte ballooning density showed an average of 57.32 cells/cm2 in the NASH diet vehicle group, 8.15
cells/cm2 in the Elafibranor group and 18.86 cells/cm2 in the OCA treatment group, which provides a more granular andquantitative measurement of the amount of hepatocyte damage than is reflected in the 0-2 CRN ballooning scoring
system (Figure 5D).

Mallory Bodies Analysis
Mallory bodies were identified and quantitated within H&E-stained liver sections as the presence of Mallory bodies within
the total image analysis area. The algorithm first identified tissue areas on whole slide images followed by positive Mallory
bodies visualized by the overlaid purple mask (Figure 6). The presence or absence of Mallory bodies is reported in tables
3&4 in the Appendix. For most of the NASH diet groups at both the 30- and 38-week timepoints, most cases have one ormore hepatocytes with Mallory-Denk bodies. The control diet group (NIH-31M) did not have ballooning hepatocytes.
​H&E NASH Diet

Mallory Body Mask NASH Diet

Figure 6. ​Mallory body analysis of NASH diet-conditioned animals at 30-weeks (A) showing liver stained with H&E left) andcorresponding purple mask, the result of cell-by-cell segmentation (right) over the same section of tissue identifying positive ballooning
cells across the entire whole slide image used for quantification.

Inflammatory Cell Analysis
Inflammatory cells were identified and quantitated within H&E-stained liver sections as the number of immune cells within
the total image analysis area. The algorithm first identified tissue areas on whole slide images followed by positive
immune cells visualized by the overlaid red mask (Figure 7A). The full data set includes immune cell density (cells/mm​2​),
total area of immune cells (mm​2​), and total number of inflammatory cells within the entire tissue area analyzed. The
number of immune cells was counted and measured as the total amount of immune cells. (Appendix Table 5&6).
The results of the inflammatory cell analysis at the 30-week timepoint showed a modest 1.2-fold increase in the immune
cell density between the control diet group (NIH-31M) and the NASH diet group (Figure 7C). At the 39-week timepoint,
this trend continued with the vehicle control NASH diet group having a 1.5-fold increase in the immune cell density overthat of the control diet group (Figure 7D). Following the 8-week treatment, the density of immune cells in the Elafibranor
treatment group did not show a significant change from that of the vehicle control NASH diet group (508.8 cells/mm​2 vs.
617.8 cells/mm​2​). 8-week OCA treatment group showed a significantly reduced immune cell density (490.8 cells/mm​2​)
compared to the levels of immune cells measured in the vehicle control NASH diet group (617.8 cells/mm​2​)(Figure 7D).

A.

B.

D.

C​.

Immune Cell Density (Week 30)

Immune Cell Density (Week 39)

E.
Inflammation Pathology Score
0 None1 1-2 foci per 20x field
2 2-4 foci per 20x field

This Study at Weeks 30, 39
Score 1-2 in NASH diet sections at 30-weeks. Score of 1 in all vehicle
control NASH diet sections at 39-weeks. At 39-week Elafibranor sections
Score 1 (4/5), Score 2 (1/5), OCA sections Score 0 (1/5) and Score 1 (4/5).
Score 0 in all control NIH31M diet sections at both 30- and 39-weeks

3 > 4 foci per 20x field

Figure 7. ​Inflammatory cell analysis of NASH diet conditioned animals at 30-weeks (A) showing liver stained with H&E (A, left) andcorresponding mask, the result of cell-by-cell segmentation (A, right) over the same section of tissue identifying immune cells (red) used
for quantification, (B) immune infiltration of lobular regions; examples of immune cell foci (blue arrows) adjacent to steatotic
hepatocytes, all NASH diet sections showed similar immune infiltration, (C) Quantitation of immune cell numbers show increased
numbers of immune cells in NASH diet animals compared to control diet (p<0.05, unpaired t-test), (D) Quantitation of immune cellnumbers at 39-weeks shows increased numbers of immune cells maintained in the NASH diet vehicle control group and that 8-weektreatment with OCA results in a significant decrease in the number of immune cells (p<0.05, unpaired t-test). Treatment with Elafibranor
does not result in a significant decrease of immune cells (p>0.05, unpaired t-test) compared to the vehicle NASH diet group, (E)Pathologist description of inflammation and score summary for control, NASH, NASH with Elafibranor, and NASH with OCA treatmentgroups.

The pathologist score for inflammation included infiltrates of leukocytes such as macrophages, lymphocytes, andneutrophils, seen primarily along the hepatic sinusoids and foci consisted of two to three leukocytes. Findings within the
NASH diet group were primarily small patchy foci within the liver lobules and no significant foci were noted in the control
diet group (CRN scores 1-2 and 0 respectively, Figure 7E)). Quantitative measure of the immune cell density (averages)
within the NASH diet group at 30-weeks was 237.0 cells/mm​2 and within the control diet group was 182.8 cells/mm​2 which
mirrors the pathologist-assigned CRN scores of 1-2 for the NASH diet group and 0 for control diet group (Figure 7C, E). At
the 39-week timepoint the averaged CRN inflammation score was 0 for the NIH-31M diet, 1 for the vehicle control NASH
diet group, 1.2 for the Elafibranor group, and 0.8 for the OCA group. The quantitated immune cell density measurements
correlate well with the average CRN inflammation scores for the control diet, vehicle NASH, NASH Elafibranor, and the
NASH OCA treatment groups (Figure 7D, E). Examination of individual animals with quantitative immune cell density
measurements gives a more in-depth, detailed assessment of incremental phenotypic changes and responses to dietinduced inflammation and treatments.

Fibrosis Analysis
For fibrosis analysis, collagen and extracellular matrix fibers within Picrosirius red-stained liver sections were identified
and quantitated as a percent of the total image analysis area. The algorithm first identified tissue areas on whole slide
images followed by positive collagen fiber regions (appear red) visualized by the overlaid green mask (Figure 8A). The
amount and intensity of collagen or fibrosis staining was measured and expressed in relation to the total image analysis
area. The full data set includes total fibrosis (%), and total amount of fibrosis within the entire tissue area analyzed (mm​2​)(Appendix Tables 7&8).
The results of the quantitative fibrosis measurement for the 30-week timepoint showed significantly more fibrosis area in
the NASH diet animals 9.30%, in comparison to 2.59% in the control (NIH-31M) diet group (p<0.001, unpaired t-test). At
the 39-week timepoint, animals fed the vehicle NASH diet had a 4.5-fold increase in the mean amount of fibrosis area
compared to animals fed the control (NIH-31M) diet. Within the treatment groups, the Elafibranor-treated group showed asignificant decrease in the mean fibrosis area (2.90%) compared to the NASH diet vehicle group (5.31%) (p<0.05,
unpaired t-test). In the OCA-treated group, the mean fibrosis area was 1.5-fold lower than the mean fibrosis area of the
NASH diet group although this was not statistically significant.

A.

B.

C.

D.
​Fibrosis (Week 30)

Fibrosis (Week 39)

E.
Fibrosis Pathology Score
0 No Fibrosis
1 Perisinusoidal or periportal
2 Perisinusoidal and periportal
3 Bridging

This Study at Weeks 30, 39
Score 1 (4/10) and 2 (6/10) in NASH diet sections at 30-weeks. Score of 2 in all
vehicle control NASH diet sections at 39-weeks. At 39-weeks Elafibranor sections
Score 1 (1/5) and Score 2 (4/5). All OCA sections Score 2 at 39-weeks. All control
NIH31M diet sections at 30-weeks score 0, at 39-weeks all score 1.

4 Cirrhosis

Figure 8. ​Fibrosis analysis of NASH diet conditioned animals at 30-weeks (A) showing liver stained with Picrosirius red (A, left) andcorresponding mask (A, right) over the same section of tissue identifying regions of fibrosis used for quantification, (B) Magnified viewof fibrotic region (stained red) showing positive perisinusoidal and periportal staining of collagen fibers, (C) Quantification of fibrosis
percentage at 30-weeks shows an increased amount of fibrosis in the NASH diet animals compared to the control diet group (p<0.001,
unpaired t-test), (D) Quantification of the amount of fibrosis at 39-weeks shows increased fibrosis in the vehicle NASH diet control
group compared to the control diet group (p<0.001, unpaired t-test). Treatment with Elafibranor significantly reduces the amount offibrosis compared to the NASH diet vehicle control group (p<0.05, unpaired t-test). Treatment with OCA does not significantly decrease
the amount of fibrosis compared to the vehicle NASH diet group, (E) Pathologist description of fibrosis and score summary for control,
NASH, NASH with Elafibranor, and NASH with OCA treatment groups.

The pathologist score for fibrosis indicates the presence of fibrosis, mainly composed of collagen, but not the quality or
thickness of the fibrous connective tissue. Within the 30-week NASH diet group 40% of the animals had periportal or
perisinusoidal fibrosis (Score 1) with 60% of the animals having periportal and perisinusoidal fibrosis (Score 2). As
expected, the AI-derived quantitative total fibrosis percentages in the NASH diet and the control diet animals correlatedwith the reported pathologist-derived fibrosis scores. The animals on the control (NIH31M) diet had no fibrosis (Score 0) at
the 30-week timepoint, and at the 39-week timepoint were reported to have mild perisinusoidal or periportal fibrosis (Score
1). The quantitative analysis of the average fibrosis percentage of the control NIH31M diet group shows a lower amount offibrosis 1.18% at the 39-week timepoint compared to the 30-week timepoint (2.59%). It is likely that the measured fibrosis
percentage does not correlate well with the pathologist-reported fibrosis scores since these scores primarily represent a
distribution or pattern of staining within the sample compared to a measurement of the amount of fibrosis. Measured
differences in the amount of average fibrosis percentages within the control diet groups at the 30- and 39-week timepoints
could be due to differences in the stain intensity, sampling variability due to the liver region, limited sample size, or may
represent actual differences in the baseline levels of collagen. At the 39-week timepoint, all of the NASH diet vehicle
animals had perisinusoidal and periportal fibrosis (Score 2). The Elafibranor-treated group had some decrease in fibrosis(20% with Score 1 and 80% with Score 2), and the OCA-treated group (all Score 2) did not show a difference from that of
the NASH diet vehicle control group (all Score 2). In contrast to the reported similar CRN fibrosis scores for NASH dietand Elafibranor treatment, the quantitative fibrosis measurement showed a significant more than 2-fold decrease in theamount of fibrosis following Elafibranor treatment (p<0.05, unpaired t-test).

A.
NASH CRN Scores for 30-Week Timepoint

B.
NASH CRN Scores for 39-Week Timepoint

Figure 9. ​Summary of NASH CRN scores for the 30-week (A) and the 39-week (B) timepoints, (A) Scores for NASH diet group are
significantly increased across all histologic features except portal inflammation in comparison to the control diet group (PSR fibrosisp<0.01, ballooning p<0.01, lobular inflammation p<0.05, steatosis p<0.0001, fibrosis p<0.0001, NAS score p<0.0001, two-way
ANOVA), (B) Scores for NASH diet vehicle group are significantly higher in comparison to the control diet group for steatosis (p<0.001),
fibrosis (p<0.0001) and the NAS score (p<0.0001)(all two-way ANOVA). The Elafibranor and the OCA treatment groups had
significantly lower fibrosis than the NASH diet vehicle control group (p<0.0001, two-way ANOVA).

Effects of NASH Treatments on Body Weights and Biomarker Analysis
The effects of the dual PPAR-α/δ agonist Elafibranor and FXR agonist Obeticholic Acid, two different stage III clinical
NASH drug treatments, were analyzed in the standard and NASH diet mice. As illustrated in Figure 10, the body weightsof the Elafibranor, OCA and NASH vehicle mice all started about 5 grams higher than that of the standard diet control
mice. Once treatment began, the NASH mouse weights responded uniquely to the treatments. Whereas the
Elafibranor-treated NASH mice lost weight and eventually weighed less than the standard diet mice at study termination,
the OCA-treated mice gained weight at a rate higher than that of the NASH diet vehicle mice (Figure 10A). Conversely,
liver weights for the Elafibranor-treated NASH mice ended up higher than those of the OCA-treated and NASH dietvehicle mice, with similar trends when liver weights were normalized to percent of body weight (Figure 10B).

A​.

B.
Effect of Treatments on Body Weights​Effect of Treatments on Liver Weights

Figure 10. ​Effect of Treatments on Body and Liver Weights of standard NIH-31M and NASH Diet Mice (A) Comparison of body weightover the course of 8-week treatment of Elafibranor- and OCA-NASH mice. At 39-weeks the NASH diet vehicle group, the Elafibranorand the OCA-treated groups showed significant increase in body weight compared to the standard diet group (p<0.001, two-way
ANOVA), (B) Comparison of liver weight at 39-weeks, the NASH diet vehicle group (p<0.0001), the Elafibranor- (p<0.0001) and the
OCA-treated (p<0.05) groups showed significant increase in liver weight compared to the standard diet mice (two-way ANOVA).

Following treatment with Elafibranor and OCA, the levels of circulating hepatic biomarkers including ALT, bilirubin,
albumin and total cholesterol were measured (Figure 11). The levels of ALT were significantly reduced in response to
Elafibranor and OCA treatment (p<0.0001 and p<0.05 respectively). Total cholesterol levels were decreased in the
Elafibranor- and OCA-treated animals in comparison to the standard diet and NASH diet mice (p<0.001 and p<0.0001
respectively). The Elafibranor and OCA treatments had no effect on the total bilirubin levels but did show some significant
increase in the amount of albumin (p<0.0001 and p<0.05 respectively).

A.

B​.
ALT

C.

Total Bilirubin

D.
AlbuminPlasma Cholesterol

Figure 11. ​Effect of NASH Therapeutics on Biomarkers for Liver Disorder and Hyperlipidemia in NASH Diet and control Mice (A) ALT
levels of NASH diet vehicle to standard control group showed significantly increased levels at both 31-weeks and 39-weeks (p<0.001
and p<0.05, two-way ANOVA), (B) Bilirubin levels of NASH diet vehicle compared to standard control group showed significantly higher
levels at 31-weeks (p<0.05, two-way ANOVA), (C) Albumin levels showed a significant increase at 39-weeks with both Elafibranor and
OCA-treatment compared to NASH diet vehicle group animals (p<0.0001 and p<0.05, two-way ANOVA) (D) Plasma cholesterol levels
at 39-weeks show increased cholesterol levels in NASH diet vehicle compared to standard control group (p<0.001, two-way ANOVA),
Elafibranor-treated (p<0.001), and OCA-treated (p<0.0001) compared to the NASH diet vehicle group (two-2-way ANOVA).

D​ISCUSSION
This study served two purposes, to characterize therelevant pathological features of the novel off-the-shelf,
diet-induced mouse NASH B6 Model (Taconic
Biosciences, Inc.) at baseline and during use in
pre-clinical drug experiments (Explora BioLabs), and todemonstrate the sensitivity and utility associated with theimageDx NASH digital assay (Reveal Biosciences). Twotimepoints were selected for analysis, including the
30-week mark to establish a baseline for measurementof the diet-induced phenotype, and then following an
8-week treatment regimen (39-week timepoint) with drugcandidates at which time steatosis, inflammation,
hepatocyte ballooning, and fibrosis were assessed.
Qualitative (human) pathology examination describednormal morphology and features in the liver sections
from the animals fed the standard control diet, but thoseanimals fed the NASH diet exhibited various disease
related abnormalities. Under the conditions of this study,
all NASH diet liver sections similarly showed prominent
steatosis with minimal to mild inflammation, minimalballooning and bile duct changes, and prominent fibrosis
in MTC-stained sections, all of which was supported by
the results of the digital quantitation assay.
Two drug candidates in phase III clinical trials were
utilized to confirm the response of diet conditioned
NASH mice to therapeutic treatment. Elafibranor, aknown dual PPAR-α/δ agonist, and documented positiveeffector of insulin sensitivity and liver injury throughanti-inflammatory and anti-fibrotic mechanisms, wasgiven to mice on the NASH diet at 30-weeks andoutcome measured at 39-weeks. The second candidate,obeticholic acid, a farnesoid X receptor (FXR) agonist,has been documented to improve insulin sensitivity andto exhibit both anti-inflammatory and anti-fibrotic
properties, as well as to improve liver biomarkers. In thisstudy, the C57BL/6NTac mice fed a high fat modified
AMLN (NASH) diet displayed increased liver and bodyweights, and high serum levels of liver diseasebiomarkers. These animals exhibited features of NASH,
including fatty liver, steatosis, inflammation, hepatocyte
ballooning, and fibrosis. Treatment of the NASH
diet-conditioned C57BL/6NTac mice with the stage III
clinical NASH drugs improved overall liver profiles. BothOCA and Elafibranor reduced hyperlipidemia/totalcholesterol and serum ALT levels. OCA treatment also
lowered liver weight in proportion to body weight.
Elafibranor possibly increased insulin sensitivity, andfrom morphological observation, reduced body weight
and fat content. Importantly, the off-the-shelf NASH B6

mice provide a cost-effective and time-saving solution for
preclinical NASH researchers, with their recapitulation of
the multi-factorial NASH phenotype and relatable
treatment responses to Elafibranor and OCA.
Throughout the study, we sought to present thequantitative results of the digital NASH assay in an
objective manner in comparison to the more subjective,
qualitative assessments of an albeit very experienced
veterinary pathologist. The subjective nature of thepathologist’s assessment of pathology is most evident in
the recording of steatosis scores. Within control diet and
NASH diet conditioned groups, both the assigned CRN
steatosis scores and the algorithm-based quantitative
results are consistent and correlate to the CRN steatosis
score, but the represented lipid percentages differ. For
example, an assigned CRN steatosis score of 3 for the
NASH diet group represents an estimated lipid
percentage greater than 67% and yet the corresponding
AI-derived percent lipid area for these same tissues ismeasured and reported at 15.1%. This difference in lipid
amount is reproducible across all studies and species todate and is likely due to both the difficulty in estimating
lipid percentages by eye and the lack of a continuous
scoring system that would allow for a more granularmeasurement similar to those generated with the
AI-derived algorithms.
The quantitative output of the imageDx NASH digitalassay provides a highly reproducible and scaled
measurement of pathological features. To assign the
CRN ballooning hepatocyte score, the pathologist isrequired to estimate the number of ballooning cells. Thistask can be hindered by the process of estimating
instead of counting the number of cells that exhibit thehallmark visual characteristics of a ballooning cell
(cytoskeletal damage and size features), and vaguenessof visual characteristics in the score label (0 none, 1 few,
2 many). One can surmise that these labels could meandifferent numbers of ballooning cells within the same
samples depending on the individual reading the slide.
Ballooning cell measurements of none, few, or many are
not granular enough to provide useful information inassessing the extent of the pathology occurring in thesamples. Some have questioned the inclusion of
ballooning hepatocyte analysis in rodent NASH models.
Interestingly, the use of the quantitative NASH digitalassay can assist with assigning a very consistentmeasurement of number of cells with positive criteria(cytoskeletal changes and large size) and could provide
a better method for not only identifying the number ofballooning cells, but also the location of these damaged
cells within the tissue and in relation to immune cell
populations.
Together, the results published within this study
contribute to the successful validation of the world’s firstcommercial, off-the-shelf mouse NASH model and thequantitative measurement of phenotype using imageDx
NASH digital assay. The development of highly
reproducible and sensitive computational models provide
an effective approach to identifying small differences intissue morphology that may not be easily discernible byvisual examination. Combined efforts included theadvanced mouse development model, ​in vivo study
management with surgeries and tissue collection, ​in vitro
analysis, pathology, histopathology, digital whole slide
scanning, and AI-based digital image analysis to quantify
fibrosis,steatosis,
inflammation,
hepatocellularballooning, and the presence of Mallory bodies. This
off-the-shelf NASH B6 rodent model successfully
recapitulates pathologic changes in NASH patients, andcombined with imageDx NASH digital assay, provides
pre-clinical researchers with an easy, reproducible,
accurate and cost effective end-to-end solution for thedevelopment of effective NASH therapeutics.

A​CKNOWLEDGMENT
The authors acknowledge the contributions of our collaborators at Taconic Biosciences, Explora BioLabs, Epredia, as wellas the expertise of Dr. Mary E.P. Goad for pathology review and scoring of animals, K. Murugesan for data analysis, and
M. Sanchez for data preparation.

A​PPENDIXQ​UANTIFICATION
Table 1. Steatosis Analysis Summary 30-Weeks (Baseline)
Data for the 30-week timepoint with the control (standard NIH-31M diet) and the NASH diet conditioned group (NASH diet) showing
total lipid area as well as more granular lipid data such as amount and type for individual hepatocytes.

* Micro droplet < 65 um^2
* Macro droplet > 65 um^2

Table 2. Steatosis Analysis Summary 39-Weeks (After Efficacy Study Conclusion)
Data for the 39-week timepoint with the control (standard NIH-31M diet), the NASH diet conditioned vehicle group (NASH diet), theElafibranor-treated NASH, and OCA-treated NASH groups showing total lipid area as well as more granular lipid data such as amountand type for individual hepatocytes.

* Micro droplet < 65 um^2
* Macro droplet > 65 um^2

Table 3. Ballooning Analysis Summary 30-Weeks (Baseline)
Data for the 30-week timepoint with the control (standard NIH-31M diet) and the NASH diet conditioned group (NASH diet) showing thedensity of ballooning hepatocytes, the tissue area analyzed (mm^2) and the presence or absence of Mallory-Denk bodies.

Table 4. Ballooning Analysis Summary 39-Weeks (After Efficacy Study Conclusion)
Data for the 39-week timepoint with the control (standard NIH-31M diet), the NASH diet conditioned vehicle group (NASH diet), theElafibranor-treated NASH, and OCA-treated NASH groups showing the density of ballooning hepatocytes, the tissue area analyzed(mm^2) and the presence or absence of Mallory-Denk bodies.

Table 5. Inflammatory Cell Analysis Summary 30-Weeks (Baseline)
Data for the 30-week timepoint with the control (standard NIH-31M diet) and the NASH diet conditioned group (NASH diet) showing thedensity of immune cells, the total number and area of immune cells, and the tissue area analyzed (mm^2).

Table 6. Inflammatory Cell Analysis Summary 39-Weeks (After Efficacy Study Conclusion)
Data for the 39-week timepoint with the control (standard NIH-31M diet), the NASH diet conditioned vehicle group (NASH diet), theElafibranor-treated NASH, and OCA-treated NASH groups showing the density of immune cells, the total number and area of immune
cells, and the tissue area analyzed (mm^2).

Table 7. Fibrosis Analysis Summary (PSR) 30-Weeks (Baseline)
Data for the 30-week timepoint with the control (standard NIH-31M diet) and the NASH diet conditioned group (NASH diet) showing thepercent fibrosis area (%), the area of total fibrosis (mm​2​), and the tissue area analyzed (mm​2​).

Table 8. Fibrosis Analysis Summary (PSR) 39-Weeks (After Efficacy Study Conclusion)
Data for the 39-week timepoint with the control (standard NIH-31M diet), the diet conditioned vehicle group (NASH diet), theElafibranor-treated NASH, and OCA-treated NASH groups showing the percent fibrosis area (%), the area of total fibrosis (mm​2​), andthe tissue area analyzed (mm​2​).

R​EFERENCES
Kleiner DE, Brunt EM, Van Natta M, Behling C, Contos MJ, Cummings OW, Ferrell LD, Liu YC, Torbenson MS,
Unalp-Arida A, Yeh M, McCullough AJ, Sanyal AJ; Nonalcoholic Steatohepatitis Clinical Research Network. Design andvalidation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology. 2005 Jun;41(6):1313-21.
Kleiner DE, Makhlouf HR. Histology of Nonalcoholic Fatty Liver Disease and Nonalcoholic Steatohepatitis in Adults andChildren. Clin Liver Dis. 2016 May;20(2):293-312.
Bedossa P and the FLIP Pathology Consortium. Fatty liver inhibition of progression (FLIP) algorithm and steatosis,
activity, and fibrosis (SAF) score in the evaluation of biopsies of nonalcoholic fatty liver disease. Hepatology. 2014
Apr;60(2):565-575.
Kleiner DE, Brunt EM, Wilson LA, Behling C, Guy C, Contos M, Cummings O, Yeh M, Gill R, Chalasani N,
Neushwander-Tetri BA, Diehl AM, Dasarathy S, Terrault N, Kowdley K, Loomba R, Belt P, Lavine JE, Sanyal AJ.
Association of histologic disease activity with progression of nonalcoholic fatty liver disease. Gastroenterology andHepatology. 2019 Oct;2(10):1-16.
Makri E, Cholangitas E, Tziomalos K. Emerging role of obeticholic acid in the management of nonalcoholic fatty liverdisease. World Journal of Gastroenterology. 2016 Nov:22(41):9039-43.
Pai RK, Kleiner DE et al. Aliment Pharmacol Ther. Standardizing the interpretation of liver biopsies in non-alcoholic fatty
liver disease clinical trials. 2019 Nov;50(10):1100-1111.
Ratziu V, Harrison SA, Francque S, Bedossa P, Lehert P, Serfaty L, Romero-Gomez M, Boursier J, Abdelmalek M,
Caldwell S, Drenth J, Anstee QM, Hum D, Hanf R, Roudot A, Megnien S, Staels B, Sanyal A. Elafibranor, an Agonist of
the peroxisome proliferator-activated receptor- α and -δ, induces resolution of nonalcoholic steatohepatitis without fibrosis
worsening. 2016 May;150 (5):1147-1159.