# US 2017/0283871 A1 · Combination of biomarkers for detecting and evaluating a hepatic fibrosis

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- Number: US 2017/0283871 A1
- Document ID: US20170283871A1
- Status: Published application
- Kind: A1
- Application number: 15/488,614
- Filed: 2017-04-17
- Published: 2017-10-05
- Earliest claimed: 2011-02-09
- Assignees: Bio-Rad Innovations; Ariana Pharmaceuticals; Institut National de la Sante et de la Recherche Medicale; Assistance Publique-Hopitaux de Paris; Centre National De La Recherche Scientifique
- Applicants: Bio-Rad Innovations; Ariana Pharmaceuticals; Institut National De La Sante Et De La Recherche Medicale; Assistance Publique - Hopitaux De Paris; Centre National De La Recherche Scientifique
- Inventors: BÉNÉDICTE WATELET; TARIK ASSELAH; IVAN BIECHE; ISABELLE CATHERINE BATXELLI; NATHALIE JULLIAN; MICHEL VIDAUD; PATRICK MARCELLIN; DANIEL LAUNE; MOHAMMAD AFSHAR; EVE LAURE MATHIEU
- Web page: https://patentgrep.com/patent/US20170283871A1

## CPC

- C12Q 1/6876: Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q 1/6883: for diseases caused by alterations of genetic material
- G01N 33/576: for hepatitis
- C12Q 1/706: {for hepatitis}
- C12Q 2600/106: Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
- C12Q 2600/112: Disease subtyping, staging or classification
- C12Q 2600/158: Expression markers
- G01N 2800/085: Liver diseases, e.g. portal hypertension, fibrosis, cirrhosis, bilirubin
- G01N 2800/60: Complex ways of combining multiple protein biomarkers for diagnosis
- G01N 2800/7052: Fibrosis
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

The application concerns means for determining the stage of hepatic tissue damage, in particular the hepatic fibrosis score of subjects infected with one or more hepatitis viruses. In particular, the means of the invention involve measuring the levels of expression of selected genes, said selected genes being: SPP1, and at least one gene from among A2M and VIM, and at least one gene from among IL8, CXCL10 and ENG, and optionally, at least one gene from among the list of the following sixteen genes: IL6ST, p14ARF, MMP9, ANGPT2, CXCL11, MMP2, MMP7, S100A4, TIMP1, CHI3L1, COL1A1, CXCL1, CXCL6, IHH, IRF9 and MMP1.

## Claims

### Claim 1

An in vitro method for determining the hepatic fibrosis score of a subject infected with one or more hepatitis viruses, characterized in that it comprises the following steps:
i) in a sample which has been obtained from said subject, measuring the levels to which the selected genes are transcribed or translated, said selected genes being:
SPP1, and
at least one gene from among A2M and VIM, and
at least one gene from among IL8, CXCL10 and ENG, and
optionally, at least one gene from among the list of the following sixteen genes: IL6ST, p14ARF, MMP9, ANGPT2, CXCL11, MMP2, MMP7, S100A4, TIMP1, CHI3L1, COL1A1, CXCL1, CXCL6, IHH, IRF9 and MMP1,
ii) comparing the measurement values of each of said selected genes obtained for said subject with their values, or with the distribution of their values, in reference cohorts which have been pre-established as a function of their hepatic fibrosis score, in order to classify said subject into that of those reference cohorts to which it has the highest probability of belonging.

### Claim 2 (depends on 1)

The method according to claim 1, in which the comparison of step ii) is carried out by combining the measurement values obtained for said subject into a multivariate classification model which compares those values with their values, or the distribution of their values, in reference cohorts which have been pre-established as a function of their hepatic fibrosis score, in order to classify said subject into that of those reference cohorts to which it has the highest probability of belonging.

### Claim 3 (depends on 1, 2)

The method according to claim 1 or 2, in which the comparison of step ii) is made by combining measurement values obtained for said subject in step i) into a pre-constructed multivariate classification model as follows:
a) for a population of individuals who are of the same species as said subject and who are infected with the same hepatitis virus or viruses as said subject, determining the hepatic fibrosis score of each of said individuals of the population, and classifying them into sub-populations as a function of their hepatic fibrosis score, thereby constituting reference cohorts established as a function of their hepatic fibrosis score;
b) in at least one sample which has already been obtained from each of said individuals the nature of which is identical to that of the sample from said subject, measuring the level of transcription or translation of each of said selected genes;
c) making an inter-cohort comparison of the measurement values obtained in step b) or the distribution of these measurement values, in order to construct a multivariate classification model which infers a hepatic fibrosis score from the combination of the levels of transcription or, if appropriate, translation of said selected genes.

### Claim 4 (depends on 1, 2, 3)

The method according to any one of claims 1 to 3, which is a method for determining whether the hepatic fibrosis of said subject has a Metavir fibrotic score of at most F1 or of at least F2, and in which said cohorts are:
a cohort of individuals with a hepatic fibrosis which has a Metavir fibrotic score of at most F1, and
a cohort of individuals with a hepatic fibrosis which has a Metavir fibrotic score of at least F2.

### Claim 5 (depends on 1, 2, 3, 4)

The method according to any one of claims 1 to 4, in which the comparison of step ii) is made by combining said measurement values obtained in step i) into a mathematical function, in particular a linear or non-linear function, more particularly a linear function, in order to obtain an output value which is indicative of the hepatic fibrosis score of said subject.

### Claim 6 (depends on 1, 2, 3, 4)

The method according to any one of claims 1 to 4, in which the comparison of step ii) is made by combining said values obtained in step i) into a multivariate machine learning model, for example a multivariate non-parametric classification model, a multivariate heuristic model, or a multivariate probabilistic prediction model, in order to obtain an output value which is indicative of the hepatic fibrosis score of said subject.

### Claim 7 (depends on 1, 2, 3, 4, 5, 6)

The method according to any one of claims 1 to 6, in which the classification of said subject into that of said reference cohorts to which it has the highest probability of belonging is made with:
a sensitivity (Se) of at least 67%, at least 68%, at least 69%, at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%, or at least 75%; and/or with a
specificity (Sp) of at least 67%, at least 68%, at least 69%, at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%, or at least 75%; and/or with
a negative predictive value (NPV) of at least 80%, or at least 81%, or at least 82%, or at least 83% or at least 84%; and/or with
a positive predictive value (PPV) of at least 50%, or at least 55%, or at least 56%, or at least 57%, or at least 58%, or at least 59% or at least 60%;
preferably with at least said NPV and/or said sensitivity, more particularly with at least said NPV and said sensitivity and said specificity.

### Claim 8 (depends on 1, 2, 3, 4, 5, 6, 7)

The method according to any one of claims 1 to 7, in which said multivariate classification model has an area under the ROC curve (AUC) of at least 0.60, at least 0.61, at least 0.66, more particularly at least 0.69, still more particularly at least 0.70, at least 0.71, at least 0.72, at least 0.73, at least 0.74, more particularly at least 0.75, still more particularly at least 0.76, still more particularly at least 0.77, in particular at least 0.78, at least 0.79, or at least 0.80.

### Claim 9 (depends on 1, 2, 3, 4, 5, 6, 7, 8)

The method according to any one of claims 1 to 8, in which said genes selected in step i) comprise A2M and/or IL8.

### Claim 10 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9)

The method according to any one of claims 1 to 9, in which:
the total number of said genes selected in step i) is four or five; and/or in which
said genes selected in step i) comprise only one or two gene(s) from said optional list of sixteen genes, or comprise none at all.

### Claim 11 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10)

The method according to any one of claims 1 to 10, in which said genes selected in step i) comprise, or are:
SPP1, A2M, IL8, CHI3L1 and IL6ST (combination No. 1); or
SPP1, A2M, IL8, ANGPT2 and IL6ST (combination No. 2); or
SPP1, A2M, IL8, IL6ST and MMP2 (combination No. 3); or
SPP1, A2M, IL8, VIM and CXCL10 (combination No. 4); or
SPP1, A2M, IL8, IL6ST and MMP9 (combination No. 5); or
SPP1, A2M, IL8, IL6ST and MMP1 (combination No. 6); or
SPP1, A2M, IL8, VIM, and ENG (combination No. 7); or
SPP1, A2M, IL8, CXCL10 and IL6ST, (combination No. 8); or
SPP1, A2M, IL8, CXCL1 and IL6ST (combination No. 9); or
SPP1, A2M, IL8 and VIM (combination No. 10); or
SPP1, A2M, IL8, COL1A1 and IL6ST (combination No. 11); or
SPP1, A2M, IL8, CXCL11 and IL6ST (combination No. 12); or
SPP1, A2M, IL8, CXCL10 and ENG (combination No. 13); or
SPP1, A2M, IL8, IL6ST and TIMP1 (combination No. 14); or
SPP1, A2M, IL8, IHH and IL6ST (combination No. 15); or
SPP1, A2M, IL8, CXCL10 and S100A4 (combination No. 16); or
SPP1, A2M, IL8, IL6ST and MMP7 (combination No. 17); or
SPP1, A2M, IL8, ENG and CXCL11 (combination No. 18); or
SPP1, A2M, ENG and MMP9 (combination No. 19); or
SPP1, A2M, CXCL10 and ENG (combination No. 20); or
SPP1, A2M, CXCL10, p14ARF and MMP9 (combination No. 21); or
SPP1, A2M, IL8, CXCL6 and IL6ST (combination No. 22); or
SPP1, A2M, IL8 and S100A4 (combination No. 23); or
SPP1, A2M, IL8, ANGPT2 and MMP7 (combination No. 24); or
SPP1, A2M, IL8, CXCL10 and p14ARF (combination No. 25); or
SPP1, A2M, IL8 and TIMP1 (combination No. 26); or
SPP1, A2M, IL8 and p14ARF (combination No. 27); or
SPP1, A2M, IL8, CXCL10 and IRF9 (combination No. 28); or
SPP1, IL8, VIM and MMP2 (combination No. 29).

### Claim 12 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11)

The method according to any one of claims 1 to 11 in which, in addition to SSP1, said genes selected in step i) comprise:
A2M, and
IL8 and/or MMP9, preferably IL8,
or
A2M and/or IL8, and
zero gene from said optional list of sixteen optional genes, or one or more genes from among said list of sixteen optional genes, wherein at least one or two genes are from among IL6ST, MMP9, S100A4, p14ARF and CHI3L1, more particularly zero gene from among said optional list of sixteen optional genes or one or more genes from among said list of sixteen optional genes, wherein at least one or two genes are from among IL6ST, MMP9 and S100A4.

### Claim 13 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)

The method according to any one of claims 1 to 12, in which said genes selected in step i) comprise, or are:
SPP1, A2M, IL8, CHI3L1 and IL6ST (combination No. 1); or
SPP1, A2M, IL8, VIM and CXCL10 (combination No. 4); or
SPP1, A2M, IL8, VIM, and ENG (combination No. 7); or
SPP1, A2M, IL8 and VIM (combination No. 10); or
SPP1, A2M, IL8, CXCL10 and ENG (combination No. 13); or
SPP1, A2M, ENG and MMP9 (combination No. 19); or
SPP1, A2M, CXCL10, p14ARF and MMP9 (combination No. 21); or
SPP1, A2M, IL8 and S100A4 (combination No. 23).

### Claim 14 (depends on 11, 12, 13)

The method according to any one of claims 11 to 13, in which:
the genes selected in step i) are the genes of one of said combination Nos. 1 to No. 29,
in step i), the levels to which each of those selected genes are transcribed are measured, and
the comparison of step ii) is made by combining said measurement values obtained in step i) into the linear Z function which is indicated for this combination of genes in Table 4, and, optionally, by comparison of the output value obtained thereby with the threshold δ indicated for this function Z in Table 5.

### Claim 15 (depends on 11, 12, 13)

The method according to any one of claims 11 to 13, in which:
the genes selected in step i) are SPP1, A2M, IL8, CXCL10 and S100A4 (combination No. 16),
in step i), the levels to which these selected genes are translated are measured, and
the comparison of step ii) is made by combining said measurement values obtained in step i) into the linear Z function which is indicated for this combination of genes in Table 6, and, optionally, by comparison of the output value obtained thereby with the threshold δ indicated for this function Z in Table 7.

### Claim 16 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)

The method according to any one of claims 1 to 15, in which:
in addition to measuring the level at which the genes selected in step i) are transcribed or translated, for said subject, the value of the following is measured, assayed or determined:
one or more clinical factor(s) such as one or more clinical factor(s) selected from: sex, age at the date of sampling, body mass index, insulin sensitivity index, diabetes, alcohol consumption, degree of steatosis, mode of contamination or Metavir activity;
and/or
one or more virological factor(s), such as one or more virological factor(s) selected from: viral genotype, duration of infection, viral load assayed for patient at treatment start date, viral load assayed for patient at sampling date; and/or
one or more biological factor(s) other than the levels of transcription or translation of said selected genes, such as one or more biological factor(s) selected from:
concentration of haptoglobin, concentration of apolipoprotein A1, total bilirubin content, concentration of gamma glutamyl transpeptidase, concentration of aspartate aminotransferase, concentration of alanine aminotransferase (ALT), platelet count, prothrombin count, quantity of cholesterol HDL, total cholesterol, concentration of ferritin, level of glycaemia, concentration of peptide C, insulin level, concentration of triglycerides, quantity of albumin, transferrin saturation, and concentration of alkaline phosphatase,
and in which
in step ii), the value(s) for this (these) factors and the measurement values for the level of transcription or translation of said genes selected in step i) are compared with their values or with the distribution of their values in reference cohorts which have been pre-established as a function of their hepatic fibrosis score in order to classify said subject into that of those reference cohorts to which it has the highest probability of belonging.

### Claim 17 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)

The method according to any one of claims 1 to 16, in which said sample which has already been obtained from said subject is:
a biological sample which has already been taken from said subject, or
a sample comprising nucleic acids and/or proteins and/or polypeptides and/or peptides extracted or purified from said biological sample, or
a sample comprising cDNA which are susceptible of having been obtained by reverse transcription of said nucleic acids,
said nucleic acids preferably being RNAs, in particular mRNAs,
said biological sample preferably being
a biological tissue or cell sample which has already been removed or collected from the liver of said subject, for example by prior hepatic biopsy puncture or by prior hepatic cytopuncture, or
a sample of biological fluid from said subject, such as a sample of blood, serum, plasma or urine.

### Claim 18 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)

A manufactured article comprising reagents in a combined preparation for their simultaneous, separate or sequential use, said reagents being constituted by:
reagents which specifically detect each of the transcription or translation products of 3 to 40 human genes, said 3 to 40 human genes comprising said selected genes according to any one of claims 1 to 17, and optionally other human genes,
and
optionally, reagents which specifically detect one or more hepatitis viruses and/or hepatitis virus genotype or genotypes.

### Claim 19 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)

A manufactured article comprising reagents in a combined preparation for their simultaneous, separate or sequential use, said reagents comprising reagents which specifically detect each of the transcription or translation products of selected genes according to any one of claims 1 to 17, in which said use is a use in a method for detecting a liver tissue damage, more particularly for detecting a hepatic fibrosis, more particularly for determining the hepatic fibrosis score of a subject, said use comprising:
taking a biological sample from said subject, in particular by inserting a needle or catheter into the body of said subject, and
using said reagents in the method according to any one of claims 1 to 17 on said biological sample, or on a sample comprising nucleic acids and/or proteins and/or polypeptides and/or peptides extracted or purified from said biological sample, or on a sample comprising cDNAs which are susceptible of having been obtained by reverse transcription of said nucleic acids.

### Claim 20 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)

A manufactured article comprising reagents in a combined preparation for their simultaneous, separate or sequential use, said reagents comprising reagents which specifically detect each of the transcription or translation products of selected genes according to any one of claims 1 to 17, in which said use is a use in a method for the treatment of a liver tissue damage of a subject, more particularly for the treatment of a hepatic fibrosis of a subject, said use comprising:
using said reagents in a method according to any one of claims 1 to 17 in order to determine the hepatic fibrosis score of said subject, and
the fact of administering to said subject a treatment aimed at blocking the progress of the hepatic fibrosis, such as standard or pegylated interferon, in a monotherapy, or in a polytherapy therapy combined with ribavirin, if the subject has a hepatic fibrosis score which, when expressed in accordance with the Metavir system, is at least F2.

### Claim 21 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)

The manufactured article according to any one of claims 18 to 20, in which said reagents hybridize specifically to the RNA of said selected genes according to any one of claims 1 to 17, and/or to the cDNA obtained by reverse transcription of these RNA, or which specifically bind to the proteins encoded by said selected genes.

### Claim 22 (depends on 18, 19, 20, 21)

The manufactured article according to any one of claims 18 to 21, in which said reagents are amplification primers and/or nucleic acid probes, or are antibodies or fragments of antibodies or aptamers.

### Claim 23 (depends on 18, 19, 20, 21, 22)

The manufactured article according to any one of claims 18 to 22, in which said reagents are contained in:
one or more tubes, or
in the wells of a nucleic acid amplification plate for receiving a sample containing nucleic acids and a reaction mixture for nucleic acid amplification, or
in the wells of a protein titration plate, more particularly a protein microtitration plate, for example an ELISA plate, or
on microbeads or on a chip for the detection and quantification of nucleic acids, proteins, polypeptides or peptides.

### Claim 24 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)

A computer program product, for storage in a memory of a processing unit or on a removable memory support for cooperation with a reader of said processing unit, characterized in that it comprises instructions for carrying out a method according to any one of claims 1 to 17.

### Claim 25 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 24)

A computer device, comprising a processing unit in the memory of which is stored a computer program product according to claim 24, and measurement values for the levels of transcription and/or translation of said selected genes as defined in any one of claims 1 to 17.