WO2013022789A2 - Compositions et procédés d'évaluation et de détermination de la présence d'un cancer du foie chez un sujet mammifère - Google Patents

Compositions et procédés d'évaluation et de détermination de la présence d'un cancer du foie chez un sujet mammifère Download PDF

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WO2013022789A2
WO2013022789A2 PCT/US2012/049621 US2012049621W WO2013022789A2 WO 2013022789 A2 WO2013022789 A2 WO 2013022789A2 US 2012049621 W US2012049621 W US 2012049621W WO 2013022789 A2 WO2013022789 A2 WO 2013022789A2
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opn
hcc
level
subject
afp
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WO2013022789A3 (fr
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Laura M. BERETTA
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Fred Hutchinson Cancer Research Center
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the invention relates to methods for determining the presence of hepatocellular carcinoma (HCC) and/or assessing the risk of developing HCC in a mammalian subject.
  • the invention relates to detection of osteopontin (OPN) levels in a biological sample obtained from a mammalian subject and comparing the detected level of OPN to a reference standard or threshold value to assess the presence of HCC, or predict the subject's risk of developing hepatocellular carcinoma prior to developing clinically detectable hepatocellular carcinoma.
  • OPN osteopontin
  • Hepatocellular carcinoma is a primary cancer of the liver. HCC is an increasingly prevalent clinical problem worldwide and is the third most common cause of cancer-related death (Venook et al, Oncologist 15(Suppl. 4):5-13, 2010). The prognosis for HCC is often poor because HCC is generally at an advanced stage of development when initially discovered. This is because HCC is usually asymptomatic at the early stages of development. Furthermore, HCC has a great propensity for intravascular invasion, even when the primary tumor is small. Therefore, relatively few hepatocellular carcinomas can be completely surgically removed once discovered.
  • HCC is often associated with chronic liver injury or disease. Cirrhosis of any etiology is the most common risk factor for HCC development. Over 90% of HCCs develop on a cirrhotic liver resulting from either chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infections, alcohol abuse, or accumulation of fat referred to as non-alcoholic steatohepatitis (NASH) (Sanyal et al, Oncologist 15(Suppl. 4): 14-22, 2010; El-Serag, Hepatol. Res. 37(Suppl. 2):S88-S94, 2007; Bugianesi, Clin. Liver Dis. 11 : 191-207, 2007).
  • HBV chronic hepatitis B virus
  • HCV hepatitis C virus
  • NASH non-alcoholic steatohepatitis
  • AFP hepatocellular carcinoma
  • AFP-L3 lectin-bound AFP
  • DCP des-gamma carboxyprothrombin
  • glypican-3 markers for HCC detection
  • the present invention provides a method for detecting the presence of hepatocellular carcinoma (HCC) in a mammalian subject at risk for developing HCC.
  • the method comprises determining the level of osteopontin (OPN) in a biological sample obtained from the subject, and comparing the OPN level to a reference standard or threshold value, wherein an elevated level of OPN compared to the reference standard or threshold value indicates the presence of HCC in the subject and the subject is classified as a candidate for treatment or further testing, and wherein an OPN level equal to or below the reference standard or threshold value indicates that HCC is not present in the subject.
  • OPN osteopontin
  • the mammalian subject at risk for developing HCC has a liver condition selected from the group consisting of cirrhosis, steatosis and fibrosis.
  • the subject is or has been infected with a hepatitis virus.
  • the subject previously received treatment for HCC.
  • the biological sample obtained from the subject is selected from the group consisting of blood, serum, plasma, urine, and liver tissue.
  • the level of OPN protein is determined by an immunoassay comprising the use of at least one antibody or antibody fragment that specifically binds to OPN protein or a portion thereof, wherein OPN has the amino acid sequence set forth in SEQ ID NO:2, 4, 6, or 22, or a naturally occurring variant thereof.
  • the level of OPN protein or a peptide fragment thereof is determined by Western blot, immuno-histological staining, ELISA assay, or mass spectroscopy.
  • the reference standard is the level of OPN determined in one or more biological samples obtained from one or more mammalian subjects of the same species that do not have HCC.
  • the reference standard value is the level of OPN determined in the subject at least once prior to the performance of step (a), wherein an increased level of OPN protein over the reference standard indicates the presence of HCC in the subject and the subject is classified as a candidate for cancer treatment or further testing.
  • the subject is undergoing treatment for HCC, wherein the reference standard value is the level of OPN determined in the subject at or prior to the initiation of the treatment, and wherein a decrease in the amount of OPN after the initiation of treatment is indicative of a positive response for the treatment.
  • the method further comprises determining the level of at least one additional marker for HCC such as alpha- fetoprotein (AFP), AFP-L3, des-y-carboxyprothrombin (DCP), lipocalin 2 (LCN2), nidogen 1 (NID-1), transmembrane glycoprotein NMB (GPNMB), and neural cell adhesion molecule (NCAM).
  • the level of the at least one additional marker is compared to a reference standard or threshold value, wherein the elevated levels of OPN and the at least one additional marker compared to the corresponding reference standard or threshold value for each marker indicates the presences of HCC in the subject.
  • the method comprises determining the level of both OPN and LCN2 in discriminating between HCC and cirrhosis.
  • the subject is a human.
  • the present invention provides a method of surveillance for the presence of hepatocellular carcinoma (HCC) in a mammalian subject.
  • the method comprises (a) determining the level of osteopontin (OPN) in a biological sample obtained from the subject; and (b) comparing the OPN level to a reference standard or threshold value, wherein an elevated level of OPN compared to the reference standard or threshold value indicates the presence of HCC in the subject and the subject is classified as a candidate for further testing or treatment, and wherein an OPN level equal to or below the reference standard or threshold value indicates that HCC is not present in the subject.
  • OPN osteopontin
  • the mammalian subject at risk for developing HCC has a liver condition selected from the group consisting of cirrhosis, steatosis and fibrosis.
  • the subject is or has been infected with a hepatitis virus.
  • the subject previously received treatment for HCC.
  • the biological sample obtained from the subject is selected from the group consisting of blood, serum, plasma, urine, and liver tissue.
  • the level of OPN is determined by measuring the level of OPN protein, or one or more peptides derived therefrom, in the biological sample.
  • the level of OPN protein is determined by an immunoassay comprising the use of at least one antibody or antibody fragment that specifically binds to OPN protein or a portion thereof, wherein OPN has the amino acid sequence set forth in SEQ ID NO:2, 4, 6, or 22, or a naturally occurring variant thereof.
  • the at least one antibody or antibody fragment is attached to or comprises a detectable label.
  • the level of OPN protein is determined by Western blot, immuno-histological staining, or ELISA assay.
  • the anti-OPN antibody or antibody fragment is attached to a solid substrate.
  • the level of OPN protein is determined by mass spectroscopy.
  • the level of OPN is determined by measuring the level of mRNA encoding the OPN protein in the biological sample.
  • the biological sample comprises liver cells.
  • the level of OPN mRNA is measured using one or more nucleic acid molecule probe(s) that specifically hybridize to at least a portion of an OPN mRNA molecule corresponding to a sequence with at least 90% homology to the cDNA sequence set forth in one of SEQ ID NOS: l, 3, 5, and 21, or the complement thereof.
  • the one or more nucleic acid molecule probes(s) is attached to or comprises a detectable label.
  • the level of mRNA is determined by Northern blot, reverse transcription quantitative polymerase chain reaction, or nuclease protection assay.
  • the reference standard is the level of OPN determined in one or more biological samples obtained from one or more mammalian subjects of the same species that do not have HCC. In some embodiments, the reference standard is the level of OPN determined in one or more biological samples obtained from one or more mammalian subjects of the same species that do not have HCC or detectable cirrhosis, steatosis or fibrosis of the liver. In some embodiments, the reference standard value is the level of OPN determined in the subject at least once prior to the performance of step (a), wherein an increased level of OPN protein over the reference standard indicates the presence of HCC in the subject.
  • the subject is undergoing treatment for HCC, wherein the reference standard value is the level of OPN determined in the subject at or prior to the initiation of the treatment, and wherein a decrease in the amount of OPN after the initiation of treatment is indicative of a positive response for the treatment.
  • the method further comprises (a) determining the level of at least one additional marker for HCC selected from the group consisting of alpha-fetoprotein (AFP), AFP-L3, des-y-carboxyprothrombin (DCP), lipocalin 2 (LCN2), nidogen 1 (NID-1), transmembrane glycoprotein NMB (GPNMB), and neural cell adhesion molecule (NCAM); and (b) comparing the level of the at least one additional marker to a reference standard or threshold value, wherein an elevated level of OPN and the at least one additional marker compared to the corresponding reference standard or threshold value for each marker indicates the presences of HCC in the subject.
  • AFP alpha-fetoprotein
  • DCP des-y-carboxyprothrombin
  • LN2 lipocalin 2
  • NID-1 nidogen 1
  • GPNMB transmembrane glycoprotein NMB
  • NCAM neural cell adhesion molecule
  • the further testing comprises at least one of a liver biopsy, liver imaging analysis, and liver enzyme functional analysis.
  • the present invention provides a method for determining a response of a mammalian subject having HCC tumor cells to treatment with an HCC inhibitory agent or surgery.
  • the method comprises: (a) determining a first level of OPN in a first biological sample taken from a mammalian subject prior to initiation of treatment with an HCC inhibitory agent or surgery; (b) determining a second level of OPN in a second biological sample taken from the mammalian subject after initiation of treatment with the HCC inhibitory agent or surgery; and (c) comparing the first and second levels of OPN, wherein a decrease in the second level of OPN measured in the second biological sample as compared to the first level of OPN measured in the first biological sample indicates a positive response to the treatment with the HCC inhibitory agent or surgery.
  • the method further comprises (a) determining a first level of at least one additional marker for HCC selected from the group consisting of alpha-fetoprotein (AFP), AFP-L3, des-y-carboxyprothrombin (DCP), lipocalin 2 (LCN2), nidogen 1 (NID-1), transmembrane glycoprotein NMB (GPNMB), and neural cell adhesion molecule (NCAM), in a third biological sample taken from a mammalian subject prior to initiation of treatment with an HCC inhibitory agent or surgery; (b) determining a second level of the at least one additional marker in a fourth biological sample taken from the mammalian subject after initiation of treatment with the HCC inhibitory agent or surgery; and (c) comparing the first and second levels of the at least one additional marker, wherein a decrease in the second level of OPN compared to the first level of OPN and a decrease in the second level of the at least one additional marker as compared to the first level of the at least one additional
  • the biological samples are plasma. In some embodiments, the subject is human.
  • the present invention provides a method for determining the risk of developing hepatocellular carcinoma (HCC) in a mammalian subject prior to the development of clinically detectable HCC.
  • the method comprises determining the level of osteopontin (OPN) in a biological sample obtained from the subject and comparing the level to a reference standard or threshold value, wherein an elevated level of OPN compared to the reference standard or threshold value indicates an increased risk that the subject will develop clinically detectable HCC.
  • OPN osteopontin
  • the method is carried out on a subject that has a liver condition selected from the group consisting of cirrhosis, steatosis and fibrosis.
  • the subject is or has been infected with a hepatitis virus.
  • the subject has previously been treated for HCC.
  • the determination of an increased risk of HCC indicates that the development of clinically detectable HCC is predicted to occur within about 24 months from the determination of the elevated OPN level in the subject.
  • the biological sample obtained from the subject is selected from the group consisting of blood, serum, plasma, urine, and liver tissue.
  • the level of OPN is determined by measuring the level of
  • the level of OPN protein is determined by an immunoassay comprising the use of at least one antibody or antibody fragment that specifically binds to OPN protein or a portion thereof, wherein OPN has the amino acid sequence set forth in SEQ ID NO:2 or a naturally occurring variant thereof.
  • the immunoassay comprises the use of at least one antibody or antibody fragment that specifically binds to an isoform of OPN resulting from alternative splicing, such as any one of the alternate OPN isoforms with the amino acid sequences set forth in SEQ ID NO:4 and 6.
  • the OPN protein has the amino acid sequence set forth in SEQ ID NO:22 or is a naturally occurring variant thereof.
  • the at least one antibody or antibody fragment is attached to or comprises a detectable label. In some embodiments, the at least one antibody or antibody fragment is attached to a solid substrate. In some embodiments, the level of OPN is determined by Western blot, immuno-histological staining, or ELISA assay. In one embodiment, an OPN protein level of 90 ng/ml of serum indicates an increased risk of developing HCC. In another embodiment, the level of OPN protein is determined by mass spectroscopy.
  • the expression level of OPN is determined by measuring the level of mRNA encoding the OPN protein, or a naturally occurring variant thereof, in the biological sample, such as a sample comprising liver cells.
  • the level of OPN mRNA is measured using one or more nucleic acid molecules primers or probes that specifically hybridize to at least a portion of an OPN mRNA molecule corresponding to a sequence with at least 90% homology to the cDNA sequence set forth in SEQ ID NO: l, 3, 5, 21, or a complement thereof, under standard hybridization conditions.
  • the one or more nucleic acid molecules is attached to or comprises a detectable label.
  • the level of mRNA is determined by Northern blot, reverse transcription quantitative polymerase chain reaction, or nuclease protection assay.
  • the reference standard is the level of OPN determined in one or more biological samples obtained from one or more mammalian subjects of the same species that do not have HCC or detectable liver disease.
  • the one or more biological samples are obtained from one or more mammalian subjects of the same species that do not have HCC or detectable cirrhosis, steatosis or fibrosis of the liver.
  • the reference standard value is the level of OPN determined at least once in the subject prior to the performance of the present method, wherein an increased level of OPN protein determined as described above over the reference standard indicates an increased risk that the subject will develop clinically detectable HCC, and the subject is a candidate for at least one of a liver biopsy, liver imaging analysis, liver enzyme functional analysis, and/or cancer treatment.
  • the method further comprises monitoring the level of OPN protein in plasma periodically obtained from the subject over time.
  • an increased level of OPN protein over time indicates the candidacy of the subject for liver biopsy, liver imaging analysis, liver enzyme functional analysis, and/or cancer treatment.
  • the method further comprises subjecting a subject identified as having an increased risk of developing HCC to an additional assay to detect and or treat cancer.
  • the method further comprises determining the level of at least one additional marker for HCC such as alpha- fetoprotein (AFP), AFP-L3, des-y-carboxyprothrombin (DCP), lipocalin 2 (LCN2), nidogen 1 (NID-1), transmembrane glycoprotein NMB (GPNMB), and neural cell adhesion molecule (NCAM).
  • AFP alpha- fetoprotein
  • DCP des-y-carboxyprothrombin
  • LN2 lipocalin 2
  • NID-1 nidogen 1
  • GPNMB transmembrane glycoprotein NMB
  • NCAM neural cell adhesion molecule
  • the invention provides a method for determining a response of a mammalian subject having HCC tumor cells to treatment with an HCC inhibitory agent or surgery, comprising: (a) determining a first level of OPN in a first biological sample taken from a mammalian subject prior to initiation of treatment with a HCC inhibitory agent or surgery; (b) determining a second level of OPN in a second biological sample taken from the mammalian subject taken after initiation of treatment with the HCC inhibitory agent or surgery; and (c) comparing the first and second levels of OPN, wherein a decrease in the second level of OPN measured in the second biological sample as compared to the first level of OPN measured in the first biological sample indicates a positive response to the treatment with the HCC inhibitory agent or surgery.
  • FIGURE 1A shows the human OPN polypeptide sequence (SEQ ID NO:2).
  • the sequences shown in bold and underlined correspond to 12 unique peptides identified by mass spectroscopy. In aggregate, these underlined and bolded sequence fragments covered 40.4% of the full length OPN polypeptide (SEQ ID NO:2), as described in Example 1;
  • FIGURE 2 graphically illustrates the OPN protein levels (ng/ml) in plasma from human patients with HCC as compared to plasma of healthy individuals (p ⁇ 0.0001), and patients with chronic hepatitis C (CHC) (p ⁇ 0.0001), or cirrhosis (p ⁇ 0.0001) from Cohort 1.
  • the box refers to the 25th and 75th percentile values with a line indicating the median levels, whereas the interquartile range extends outside the box. Points outside the interquartile range are outliers, as described in Example 2;
  • FIGURE 3A shows a receiver operating characteristic (ROC) curve evaluating the abilities of the AFP and OPN markers, or the combination thereof, to distinguish between individuals in Cohort 1 with HCC and individuals in Cohort 1 with cirrhosis.
  • the area under the ROC curve (AUC) is shown with the 95% confidence intervals; AFP is represented by the solid line, OPN is represented by the dashed line, and the combination of AFP and OPN is represented by the dotted line; as described in Example 2;
  • FIGURE 3B shows a receiver operating characteristic (ROC) curve evaluating the abilities of the AFP and OPN markers, or the combination thereof, to distinguish between individuals in Cohort 1 with HCV-related HCC and individuals in Cohort 1 with HCV-related cirrhosis.
  • the area under the ROC curve (AUC) is shown with the 95% confidence intervals; AFP is represented by the solid line, OPN is represented by the dashed line, and the combination of AFP and OPN is represented by the dotted line; as described in Example 2;
  • FIGURE 3C shows a receiver operating characteristic (ROC) curve evaluating the abilities of the AFP and OPN markers, or the combination thereof, to distinguish between individuals in Cohort 1 with early stage (Barcelona stages 0 or A) HCC and individuals with cirrhosis.
  • the area under the ROC curve (AUC) is shown with the 95% confidence intervals; AFP is represented by the solid line, OPN is represented by the dashed line, and the combination of AFP and OPN is represented by the dotted line; as described in Example 2;
  • FIGURE 3D shows a receiver operating characteristic (ROC) curve evaluating the abilities of the AFP and OPN markers, or the combination thereof, to distinguish between individuals in Cohort 1 with HCC (and with AFP levels below 20 ng/ml) and individuals in Cohort 1 with cirrhosis.
  • the area under the ROC curve (AUC) is shown with the 95% confidence intervals; AFP is represented by the solid line, OPN is represented by the dashed line, and the combination of AFP and OPN is represented by the dotted line; as described in Example 2;
  • FIGURE 4A graphically illustrates OPN protein levels (ng/ml) in plasma from human patients with HCC as compared to OPN levels in healthy individuals (p ⁇ 0.0001), and OPN levels in patients with cirrhosis or chronic HBV (CHB) (p ⁇ 0.001) from Cohort 2.
  • the box refers to the 25th and 75th percentile values with a line indicating the median levels, whereas the interquartile range extends outside the box. Points outside the interquartile range are outliers, as described in Example 2;
  • FIGURE 4B shows a receiver operating characteristic (ROC) curve evaluating the abilities of the AFP and OPN markers, or the combination thereof, to distinguish between individuals in Cohort 2 with HCC and individuals in Cohort 2 with chronic hepatitis B (CHB) and cirrhosis.
  • ROC receiver operating characteristic
  • AUC area under the ROC curve
  • FIGURE 4C shows a receiver operating characteristic (ROC) curve evaluating the abilities of the AFP and OPN markers, or the combination thereof, to distinguish between individuals in Cohort 2 with HBV-related HCC and individuals in Cohort 2 with HBV -related cirrhosis and/or chronic HBV (CHB).
  • ROC receiver operating characteristic
  • AUC area under the ROC curve
  • FIGURE 4D shows a receiver operating characteristic (ROC) curve evaluating the abilities of the AFP and OPN markers, or the combination thereof, to distinguish between individuals in Cohort 2 with HCC and AFP levels below 20 ng/ml and individuals in Cohort 2 with HBV-related cirrhosis and/or chronic HBV (CHB).
  • ROC receiver operating characteristic
  • AUC area under the ROC curve
  • FIGURE 5A graphically illustrates the OPN protein (ng/ml) levels and AFP protein (ng/ml) levels in plasma from samples obtained from 22 patients at the time of HCC diagnosis.
  • the horizontal and vertical dotted lines indicate the threshold cutoff for prediction of HCC based on OPN or AFP, respectively; as described in Example 3;
  • FIGURE 5B graphically illustrates the OPN protein (ng/ml) levels and AFP protein (ng/ml) levels in plasma from samples obtained from the same 22 patients analyzed in the graph shown in FIGURE 5 A at a time period of 6 to 12 months prior to HCC diagnosis.
  • the horizontal and vertical dotted lines indicate the threshold cutoff for prediction of HCC based on OPN or AFP, respectively; as described in Example 3;
  • FIGURE 5C graphically illustrates the OPN protein (ng/ml) levels and AFP protein (ng/ml) levels in plasma from samples obtained from the same 22 patients analyzed in the graph shown in FIGURE 6 A at a time period of 12 to 24 months prior to HCC diagnosis.
  • the horizontal and vertical dotted lines indicate the threshold cutoff for prediction of HCC based on OPN and AFP, respectively; as described in Example 3;
  • FIGURE 5D graphically illustrates the OPN protein (ng/ml) levels and AFP protein (ng/ml) levels in plasma from samples obtained from the same 22 patients analyzed in the graph shown in FIGURE 6A at a time period of 24 to 48 months prior to HCC diagnosis.
  • the horizontal and vertical dotted lines indicate the threshold cutoff for prediction of HCC based on OPN and AFP, respectively; as described in Example 3;
  • FIGURE 6A graphically illustrates OPN splice variant isoform B protein levels in plasma from human patients with HCC compared to plasma of human patients with cirrhosis.
  • OPN isoform B protein abundance is shown as the total number of tandem mass spectra assigned to that protein in each sample; as described in Example 4;
  • FIGURE 6B graphically illustrates the fold change of mRNA for OPN splice variant isoforms A, B, and C in human HCC tumors compared to a pool of normal liver tissue; as described in Example 4;
  • FIGURE 7A graphically illustrates OPN protein abundance in HCC liver tissue compared to liver tissue from control mice and Pten null mice with NASH. The abundance is shown as normalized peptide hits identified by mass spectroscopy, as described in Example 5;
  • FIGURE 7B graphically illustrates OPN protein abundance in plasma from 9 and 12 month old Pten null mice with HCC compared to the plasma from control mice and 6 month old Pten null mice with NASH. The abundance is shown as normalized peptide hits identified by mass spectroscopy, as described in Example 5;
  • FIGURE 7C graphically illustrates LCN2 protein abundance in HCC liver tissue from Pten null mice compared to liver tissue from control mice and Pten null mice with NASH. The abundance is shown as normalized peptide hits identified by mass spectroscopy, as described in Example 5;
  • FIGURE 7D graphically illustrates LCN2 protein abundance in plasma from 9 and 12 month old Pten null mice with HCC compared to the plasma from control mice and 6 month old Pten null mice with NASH. The abundance is shown as normalized peptide hits identified by mass spectroscopy, as described in Example 5;
  • FIGURE 8A graphically illustrates the fold change of OPN mRNA levels in HCC tumor tissue, liver tissue adjacent to the tumor, and liver tissue from mice with NASH, compared to control liver, as determined by quantitative RT-PCR.
  • the * indicates p ⁇ 0.01, as described in Example 5;
  • FIGURE 8B graphically illustrates OPN protein abundance in plasma from Pten null mice with HCC compared to the plasma from control mice and Pten null mice with NASH. The abundance was determined by ELISA. The * indicates p ⁇ 0.01, as described in Example 5;
  • FIGURE 8C graphically illustrates the fold change of LCN2 mRNA levels in HCC tumor tissue, liver tissue adjacent to the tumor, and liver tissue from mice with NASH, compared to control liver, as determined by quantitative RT-PCR.
  • the * indicates p ⁇ 0.01, as described in Example 5;
  • FIGURE 8D graphically illustrates LCN2 protein abundance in plasma from Pten null mice with HCC compared to the plasma from control mice and Pten null mice with NASH. The abundance was determined by ELISA. The * indicates p ⁇ 0.01, as described in Example 5;
  • FIGURE 9A and B graphically illustrate temporal increase in OPN and LCN2 protein levels in plasma collected prospectively in one control mouse and two Pten null mice, as described in Example 5;
  • FIGURE 10A and B graphically illustrate the increase in serum levels of OPN and LCN2, respectively, from cirrhosis patients (Cirr) and hepatocellular carcinoma patients (HCC), as described in Example 7; and
  • FIGURE 11A, 11B, 11C, and 11D graphically illustrate the ROC curve analysis for each marker, where FIGURE 11 A depicts AFP, FIGURE 1 IB depicts DCP, FIGURE l lC depicts OPN, and FIGURE 1 ID depicts LCN2, as described in Example 7.
  • FIGURE 11 A depicts AFP
  • FIGURE 1 IB depicts DCP
  • FIGURE l lC depicts OPN
  • FIGURE 1 ID depicts LCN2, as described in Example 7.
  • the term "about” refers to a range of variation that extends above or below the stated value by an amount of 10% of the stated value. As an example, the phrase “about 50” is intended to encompass values ranging between and including 45 and 55.
  • HCC hepatocellular carcinoma tumors in the liver that are detectable through the application of accepted diagnostic techniques.
  • HCC diagnostic guidelines are provided, for example, by the American Association for the Study of Liver Diseases (AASLD) (5).
  • AASLD American Association for the Study of Liver Diseases
  • HCC tumors can be detected by the application of non-invasive liver imaging tests, which include CT scans, abdominal ultrasound, and magnetic resonance imaging.
  • MRI and CT scans for example, use contrast agents that are injected into the body and enhance the visibility of the structural features of the liver.
  • the imaging analyses facilitates the detection of lesions on the liver tissue. These data can be complemented using liver function enzyme testing, known in the art.
  • the term "prior to developing clinically detectable HCC” refers to pre-HCC diagnosis period during which standard techniques to detect HCC, described above, are not able to detect the presence of hepatocellular carcinoma cells or tumors in the liver.
  • pre-HCC diagnosis period the subject would be negative for liver lesions as determined by ultrasonography.
  • the term “early HCC” refers to early stages of clinically detectable hepatocellular carcinoma. Specifically, the early stage of HCC is defined as "Barcelona stage 0 and A”, according to AASLD guidelines.
  • chronic hepatitis refers to the persistent inflammation of the liver cells. Hepatitis is generally considered “chronic” when the inflammation lasts for a period of more than 6 months. While chronic hepatitis can be asymptomatic, it often leads to jaundice, loss of appetite, and malaise. Viral infection of liver cells by viruses such as hepatitis A, B, and C, is most often the cause of chronic hepatitis, although it can also be caused by exposure to toxins and autoimmune reactions.
  • steatosis refers to the abnormal retention of lipids within a cell. Because the liver is the primary organ involved in lipid metabolism, the liver is most often associated as the primary location of steatosis. While early stages of steatosis are often asymptomatic, inflammation can occur, often leading to symptoms such as fatigue and weight loss.
  • fibrosis refers to the accumulation of scar tissue in the liver, which often leads to the reduction of functional liver tissue, and ultimately loss of liver function. Fibrosis is often caused by infections, accumulation of fat, or prolonged exposure to toxins, such as alcohol.
  • cirrhosis refers to a condition of the liver resulting from extensive or advanced fibrosis that results in significant loss of liver function.
  • the term "positive response to the treatment of HCC in the mammalian subject” refers to a reduction in tumor volume and/or inhibition of tumor growth after treatment with an HCC inhibitory agent.
  • HCC inhibitory agent is any agent, such as a small molecule, protein therapeutic, antibody or nucleic acid molecule, that causes growth inhibition of HCC tumor cells either in vitro in cultured cells and/or in vivo in a mammalian subject.
  • Alpha-fetoprotein is utilized worldwide as a surveillance biomarker for HCC.
  • the performance characteristics of AFP as a diagnostic and screening test for HCC are limited. AFP performance characteristics are particularly limited in patients with HCV and in early HCC. This was confirmed in the results obtained in Cohort 1, as described below in Examples 2 and 3.
  • Patients with HCC or cirrhosis have higher levels of circulating osteopontin "OPN" than non-HCC subjects in a study using comparative proteomic profiling of plasma, as described below in Example 1.
  • OPN is a secreted phosphoprotein that binds the integrin alpha V beta and CD44 families of receptors (El-Tanani, Front. Biosci. 13:4276-4284, 2008).
  • Genbank accession number for the polypeptide sequence of the canonical human OPN (isoform A) is NP 001035147, and the amino acid sequence is set forth as SEQ ID NO:2.
  • Genbank accession number for the cDNA corresponding to the mR A transcript encoding the above OPN polypeptide is NM OO 1040058.1, and the nucleotide sequence is set forth as SEQ ID NO: l .
  • OPN also encompasses naturally occurring variants of the canonical form of OPN, such as polypeptide molecules resulting from alternative or differential splicing of the messenger RNA for OPN.
  • the amino acid sequences for the isoform B variant of OPN is set forth herein as SEQ ID NO:4, encoded by the nucleotide sequence set forth herein as SEQ ID NO:3.
  • the amino acid sequences for the isoform C variant of OPN is set forth herein as SEQ ID NO:6, encoded by the nucleotide sequence set forth herein as SEQ ID NO:5.
  • OPN also encompasses any mammalian homo log of any one of the human peptides mentioned above, for instance mouse OPN protein, the sequence of which is set forth herein as SEQ ID NO:22, encoded by the nucleic acid sequence set forth herein as SEQ ID NO:21.
  • Natural variants of these OPN sequences also include sequences having at least 90%, 95%, 96%, 97,%, 98%, and 99% identity to SEQ ID NOS: l-6, and 21-22.
  • OPN OPN was confirmed as a useful biomarker for HCC that outperformed the standard AFP marker in distinguishing HCC patients from control at-risk patients, such as those with cirrhosis and hepatitis virus infections, in two cohorts with distinct disease etiologies.
  • OPN was significantly more sensitive than AFP for the diagnosis of HCC in all studied HCC groups.
  • OPN performed well in detecting AFP -negative HCC.
  • OPN performed particularly well in patient groups for which AFP performance was poor, such as in HCV patients and patients with early stages of HCC.
  • the best marker performance was the combination of AFP and OPN indicating that these two markers are complementary.
  • OPN performed well in combination with additional markers which are found to be good predictors for the presence of HCC.
  • the additional markers are lipocalin 2 (LCN2), nidogen 1 (NID-1), transmembrane glycoprotein NMB (GPNMB), and neural cell adhesion molecule (NCAM).
  • Example 3 elevated levels of OPN were detected in plasma samples collected up to 24 months in "at-risk" patients at a time when patients were deemed negative for HCC by standard diagnostic techniques and before the eventual clinical diagnosis of HCC. In contrast, AFP levels for the majority of these patients were not elevated.
  • splice variant isoforms of OPN can also serve as biomarkers for the presence or onset of HCC in humans.
  • a murine model is used to demonstrate that levels biomarkers OPN and LCN2 also correspond with the progression of HCC tumors, and thus are useful for monitoring the stage of disease and can be applied to assess the response to treatment.
  • the present invention provides a method for determining the likelihood of development of hepatocellular carcinoma (HCC) in a mammalian subject prior to the onset of clinically detectable HCC.
  • HCC hepatocellular carcinoma
  • OPN is used as a biomarker for risk stratification, in which the subject is assessed for the relative risk of developing HCC.
  • the assessment at risk can be made at a time when no HCC is clinically detectable, such as by ultrasound.
  • the method comprises determining the level of osteopontin (OPN) in a biological sample obtained from the subject, and comparing the level of OPN to a reference standard or threshold value, wherein an elevated level of OPN compared to a reference standard or threshold value indicates a higher risk of HCC onset in the subject than a subject that has a level of OPN below the reference standard or threshold value.
  • OPN osteopontin
  • a current advantage of the present method is that elevated levels of OPN serve as a biomarker for predicting the likelihood of developing clinically detectable HCC within a period of time after performance of the method.
  • the method is performed on a biological sample obtained from a subject that does not currently have clinically detectable HCC.
  • clinically detectable HCC refers to the presence of primary hepatocellular carcinoma cells or tumors in the liver that are detectable through the application of accepted diagnostic techniques. Such techniques are known in the art, and preferably include AFP detection, liver imaging, such as through CT scans and MRI, and/or the performance of a liver enzyme functional analysis.
  • the method allows for the determination of the likelihood of development of HCC and/or the detection of the presence of HCC. Based on the determined level of OPN in the biological sample obtained from a subject relative to a reference standard or threshold level, the subject can be classified as having HCC, or as high risk or low risk of future HCC onset. In one embodiment, a subject that is high risk is more likely than not to develop HCC following the determination of OPN levels in the biological sample.
  • the risk can be expressed in terms of percent risk. For example, in one embodiment, a subject with a high risk of HCC onset has more than about a 50% chance of developing HCC. In further embodiments, the risk can be expressed as more than about 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or higher.
  • the risk of HCC onset in the subject can be expressed in terms of a period of time following the determination of OPN levels in the biological sample.
  • the risk of development of HCC in the subject can be determined for the 24 months following the determination of OPN levels in the biological sample obtained from the subject.
  • the indicated risk can be determined for periods such as about 24 22, 20, 18, 16, 14, 12, 10, 8, 6, and 4 months following the determination of OPN levels in the biological sample obtained from the subject. For example, as described below in Example 3, high levels of OPN were detected in human subjects between 6 months and 24 months before the actual clinical detection of HCC.
  • the development of HCC is often associated with pre-existing liver conditions and diseases.
  • Subjects with diseases such as steatosis, fibrosis, cirrhosis, hepatitis, and/or infection with hepatitis viruses, are considered "at-risk" for HCC because they are more likely than healthy subjects to develop HCC. Therefore, in some embodiments, the method is performed to determine the likelihood of development of HCC in mammalian subjects that are "at-risk" for HCC, but at a time that is prior to the onset of clinically detectable HCC.
  • the subject has a liver condition, such as cirrhosis, steatosis, fibrosis, or hepatitis.
  • the subject is or has been infected with a hepatitis virus, such as hepatitis A, B, or C virus, including chronic infections.
  • the subject has been previously diagnosed with, and received or is currently receiving treatment for HCC.
  • a subject may have received treatment for HCC and subsequently been determined to be negative for the presence of clinically detectable HCC.
  • the subject is monitored periodically for the risk of recurrence of clinically detectable HCC by measuring the level of OPN in a biological sample obtained from the subject in accordance with the methods described herein.
  • the subject is undergoing treatment for HCC or may be receiving prophylactic treatment against the recurrence of HCC or other cancers.
  • the subject can be monitored for the risk of developing clinically detectable HCC.
  • a rise or fall of detected OPN levels can be an indicator of the efficacy or efficiency of the treatment over time.
  • a person of ordinary skill in the art will recognize that many subjects have multiple liver conditions, the incidence of which can be related. Therefore, in some embodiments the subject has a combination of liver conditions, including those described above.
  • An "at-risk" subject with any pre-existing liver condition or disease, or a prior history of a liver condition or disease can be subjected to one or more applications of the methods described herein to monitor the subject for the risk of developing clinically detectable HCC.
  • OPN is generally a secreted protein.
  • the method of the present invention can be performed on any biological sample that contains secreted proteins.
  • the biological sample a biological fluid such as whole blood or a fluid derived from whole blood.
  • the biological sample can be another bodily fluid, such as urine.
  • the biological sample contains liver cells.
  • the biological sample can be liver cells obtained by biopsy.
  • OPN OPN-induced transcriptional activity of the cell
  • the transcriptional state of OPN can be conveniently determined by measuring transcript abundance by any of several existing gene expression technologies that are well-known in the art.
  • the translational state includes the identities and abundance of the constituent polypeptide species in the sample generated by the ribosomal machinery using the mRNA template described above.
  • the translational state i.e., level of constituent OPN polypeptide, can also be conveniently determined by various technologies that are well-known in the art.
  • the level of OPN gene expression can be measured by amplifying OPN messenger RNA obtained from the cells using reverse transcription (RT) in combination with the polymerase chain reaction (PCR). Briefly, total RNA, or mRNA from a sample is used as a template, and a primer specific to mRNA polyA tail, or to the transcribed portion of the OPN gene (see SEQ ID NO: l for a human cDNA sequence of OPN) is used to initiate reverse transcription. Methods of reverse transcribing RNA into cDNA are well known and described in Sambrook et ⁇ , 1989, supra. The product of the reverse transcription is subsequently used as a template for PCR using template specific primers. The method of PCR and primer design is well known in the art.
  • the PCR method is quantitative RT PCR (qRT PCR or qPCR), to more precisely quantify OPN mRNA levels, and thus the OPN gene expression levels.
  • qRT PCR quantitative RT PCR
  • qPCR is performed with a transcript-specific antisense probe.
  • a PCR is prepared with a quencher and fluorescent reporter probe complexed to the 5' end of the oligonucleotide.
  • Taq DNA polymerase When Taq DNA polymerase is activated, it cleaves off the fluorescent reporters of the probe bound to the template by virtue of its 5' to 3' exonuclease activity.
  • the reporters now fluoresce providing a quantifiable signal to ascertain template abundance at every round of amplification.
  • An alternative technique is to use an intercalating dye such as the commercially available QuantiTectTM SYBR® Green PCR (Qiagen, Valencia, CA). RT-PCR is performed using SYBR® green as a fluorescent label which is incorporated into the PCR product during the PCR stage and produces a fluorescence proportional to the amount of PCR product.
  • nuclease protection assays can be used to detect and quantitate OPN mRNA.
  • an antisense probe (labeled with, e.g., radiolabeled or nonisotopic label) hybridizes in solution to an RNA sample. Following hybridization, single-stranded, unhybridized probe and RNA are degraded by nucleases. An acrylamide gel is used to separate the remaining protected fragments.
  • solution hybridization is more efficient than membrane-based hybridization, and it can accommodate up to 100 ⁇ g of sample RNA, compared with the 20-30 ⁇ g maximum of blot hybridizations.
  • the ribonuclease protection assay which is the most common type of nuclease protection assay, requires the use of RNA probes. Oligonucleotides and other single-stranded DNA probes can only be used in assays containing SI nuclease.
  • the single-stranded, antisense probe must typically be completely homologous to the target RNA to prevent cleavage of the probe-target hybrid by the nuclease.
  • the antisense probe can correspond to any section of the OPN mRNA sequence (see SEQ ID NO: 1 for a human OPN cDNA transcript corresponding to the mRNA).
  • RNA samples are obtained and separated by size via electrophoresis.
  • the RNA is transferred to a membrane, crosslinked and hybridized with a labeled probe that hybridizes specifically to the target mRNA under defined hybridization conditions.
  • the probe can be labeled by any of the many different methods known to those skilled in this art. The labels most commonly employed for these studies are radioactive elements, enzymes, chemicals that fluoresce when exposed to ultraviolet light, and others.
  • RNA need not be extracted from the sample.
  • fluorescent in situ hybridization can be used to determine the presence, relative quantity, and spatial distribution of target mRNA in a cell.
  • Single Molecule RNA FISH Biosearch Technologies, Novato, CA
  • Single Molecule RNA FISH uses multiple short, singly-labeled oligonucleotide probes complementary to distinct portions of the target sequence. When each probe binds to the single stranded mRNA template, it causes cooperative unwinding of the mRNA, promoting the binding of the additional probes. The net result is the binding of a large multitude of fluorescent labels to a single molecule of mRNA template, providing sufficient fluorescence to reliably locate each target mRNA in a wide-field fluorescent microscopy image.
  • the gene expression level can be determined by assessing the translational state of the OPN gene.
  • standard techniques can be used for determining the amount of the OPN polypeptide or protein present in a sample. For example, immunoassays such as Western blot involve immunoprecipitation of protein from a sample according to methods well-known in the art. This is followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the protein sample. After separation of the proteins, immunocytochemistry and the like can be used to determine the amount of the protein or proteins of interest present in a sample.
  • a typical agent for detecting a protein of interest is a detectable antibody, or fragment thereof, capable of binding to the protein of interest.
  • antibodies, or fragments thereof can be employed to detect levels of OPN polypeptide, including splice variant isoforms, in a sample by techniques such as with an enzyme-linked immunosorbent assay (ELISA). Briefly, an antibody or fragment thereof that specifically binds to a portion of the OPN polypeptide is immobilized on a solid substrate. The sample is contacted to the solid substrate and any OPN polypeptides present in the sample are permitted to bind to the immobilized (capture) antibody. A second antibody that also binds to the OPN polypeptide is also contacted with the biological sample and/or solid substrate and permitted to bind to the OPN polypeptide. The second antibody usually comprises a detectable label or signal. The label can be detected directly, or it may be a component of a signal-generating system, which are well-known in the art. The level of OPN is determined by assaying the detectable signal after the solid substrate has been rinsed of unbound sample and antibody.
  • ELISA enzyme-linked immunosorbent assay
  • antibodies, or fragments thereof can be employed histologically, as in immunofluorescence or immunoelectron microscopy, for in situ detection of a protein of interest.
  • In situ detection can be accomplished by obtaining a histological specimen (e.g., a biopsy specimen or immobilized cell culture) and applying thereto a labeled antibody that is directed to the target protein.
  • the antibody (or fragment) is preferably applied onto a biological sample, such as liver cells from a section or biopsy.
  • a biological sample such as liver cells from a section or biopsy.
  • Antibodies can be generated utilizing standard techniques well known to those of skill in the art. Such antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or an antibody fragment (e.g., Fab or F(ab')2), that specifically hybridizes to a portion of the OPN polypeptide can be used.
  • an antibody fragment e.g., Fab or F(ab')2
  • an enzyme bound to the antibody will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fiuorometric or by visual means.
  • the detection can be accomplished by colorimetric methods which employ a chromogenic substrate for the enzyme.
  • Detection can also be accomplished by radioactively labeling the antibodies or antibody fragments, and detecting the protein of interest through the use of a radioimmunoassay. It is also possible to label the antibody with a fluorescent compound which fluoresces upon stimulation by light of a proper wavelength.
  • the determined likelihood of development of clinically detectable HCC is based on the comparison of detected OPN levels in a biological sample obtained from the subject with a reference standard.
  • the reference standard is the OPN level in a sample or samples obtained from one or more individuals of the same species that have been determined to not have HCC or have been determined to not be at-risk of having HCC.
  • the individual(s) used as reference standards are healthy, or at least do not have any detectable liver conditions.
  • One or more, including 2, 3, 4, 5, 10, or more individuals, can be used to generate a reference standard level of OPN.
  • the OPN levels for the individuals can be averaged to create a single value.
  • the reference standard is an established threshold level.
  • the threshold level can be determined, for example, by using the maximum sum of sensitivity and specificity, or alternatively, using the minimum distance to the top-left corner of the ROC curve, as described below in Example 2.
  • the determined clinical cutoff threshold levels for OPN were determined in Example 2 to be 91 ng/ml and 149 ng/ml for Cohort 1, and 156 ng/ml for Cohort 2.
  • the reference standard is an OPN level obtained from the subject at a prior time point.
  • the methods described herein are performed multiple times on the subject over a period of time to monitor for increasing OPN levels, which would indicate increasing risk of development of clinically detectable HCC.
  • the method described herein is useful for determining the likelihood of development of HCC in mammalian subjects.
  • the method includes determination of risk of development of HCC in rodents, primates, cows, horses, dogs, cats, and other mammals.
  • the mammalian subject is a rat, mouse, or human.
  • a useful feature of the present invention is that the development of clinically detectable HCC can be predicted prior to the actual development thereof. As a result, the potential presence of HCC can be addressed while still in the very early stages in liver disease. Therefore, the present invention is useful for identifying subjects in need of additional or more intensive monitoring or surveillance.
  • the subject is pre-determined to be "at-risk" or predisposed to HCC due to existing non-cancerous liver disease, such as cirrhosis, fibrosis, steatosis or hepatitis.
  • the present method may be performed multiple times, such as every month, 3 months, 6 months, or year to monitor the "at-risk" subject for changes in OPN levels.
  • the OPN levels are compared to a reference standard to determine the risk of developing clinically detectable HCC, according to the principles of the present method.
  • the initial determined level of OPN in a sample obtained from a subject serves as the reference standard level.
  • OPN levels from biological samples subsequently obtained from the subject are compared to the initial OPN level. Elevated levels of OPN in the subsequently obtained samples indicates an increased risk of developing clinically detectable HCC. Based on this indication, the subject can be considered a candidate for additional HCC diagnostic tests, such as liver imaging analyses, liver enzyme functional analysis, and/or liver biopsy.
  • the subject can be considered a candidate for early cancer intervention treatments, such as surgery (e.g., hepatic resection and liver transplantation), chemotherapy (e.g., transcatheter arterial chemoembolisation), and local ablative therapy, such as radio frequency ablation or percutaneous ethanol injection.
  • surgery e.g., hepatic resection and liver transplantation
  • chemotherapy e.g., transcatheter arterial chemoembolisation
  • local ablative therapy such as radio frequency ablation or percutaneous ethanol injection.
  • the present invention is useful for monitoring a subject previously treated for HCC, or currently undergoing treatment, for recurrence thereof.
  • the subject can be periodically monitored by performance of the methods described herein to ascertain the risk of recurrence of clinically detectable HCC.
  • Any increase in OPN levels compared to a reference standard indicates an increased risk that the subject will have a recurrence of HCC.
  • an increase in OPN levels in a biological sample obtained from the subject previously treated for HCC compared to HCC levels previously determined from the subject indicates an increased risk that the subject will have a recurrence of HCC.
  • the reference standard level of OPN is determined in a biological sample obtained from the subject after the prior treatment for HCC.
  • the present method also encompasses the monitoring or assessment of other known indicators of HCC, in addition to, or combination with the determination of OPN levels, as described herein.
  • levels of other biological markers for HCC such as AFP
  • AFP may also be determined in a biological sample obtained from the subject.
  • Other biomarkers useful in this aspect include AFP, lipocalin 2 (LCN2), nidogen 1 (NID-1), transmembrane glycoprotein NMB (GPNMB), and neural cell adhesion molecule (NCAM), as described below in Examples 1 and 2, and AFP-L3 and des-Y-carboxyprothrombin (DCP).
  • the biological sample may be the same or different from the biological sample used to determine the level of OPN, as described above.
  • the levels of other biological marker(s) may be determined in the same or different assay as used to determine the level of OPN.
  • a multiplex assay may be used to determine the levels of OPN and AFP in the same biological sample obtained from the subject.
  • the levels of OPN and AFP, or any other marker for HCC such as AFP-L3, des-Y-carboxyprothrombin (DCP), LCN2, NID-1, GPNMB, and NCAM, can be determined in the same or different biological sample, in an independent assay.
  • the indicator of HCC can be data obtained from visualization of the liver, such as by ultrasound, CT scan, and MRI, wherein observed lesions can be indicative of HCC.
  • the additional indicator of HCC may be obtained from a liver enzyme functional assay.
  • the present invention provides for methods of detecting the presence of HCC in a mammalian subject at risk of developing HCC.
  • the method comprises determining the level of osteopontin (OPN) in a biological sample obtained from the subject, and comparing the OPN level to a reference standard or threshold value, wherein an elevated level of OPN compared to the reference standard or threshold value indicates the presence of HCC in the subject and the subject is classified as a candidate for treatment or further testing, and wherein an OPN level equal to or below the reference standard or threshold value indicates that HCC is not present in the subject.
  • OPN osteopontin
  • Subjects "at risk” for developing HCC have been described and include subjects with liver conditions, such as cirrhosis, steatosis, fibrosis, prior infections with hepatitis viruses, and prior treatment for HCC.
  • the method can be performed on biological samples using immunoassay techniques (e.g., ELISA), mass spectroscopy, and techniques to quantify gene expression.
  • the method is further supplemented by also assaying for changes in levels of other biomarkers, such as AFP, AFP-L3, des-Y-carboxyprothrombin (DCP), LCN2, NID-1, GPNMB, and NCAM, or the performance of imaging analysis, such as with ultrasonography.
  • biomarkers such as AFP, AFP-L3, des-Y-carboxyprothrombin (DCP), LCN2, NID-1, GPNMB, and NCAM, or the performance of imaging analysis, such as with ultrasonography.
  • the present invention provides a method of surveillance for the presence of HCC in a mammalian subject.
  • the methods, compounds, and techniques described herein can be applied to screen subjects for early HCC for which intervention is possible.
  • the level of OPN is determined in a biological sample from the subject and compared to a reference or threshold value, as previously described. An elevated level of OPN in the sample indicates the presence of HCC in the subject.
  • the method of surveillance is useful, for instance, in monitoring or screening patients deemed to be "at risk" of developing HCC to facilitate early intervention upon detection of HCC.
  • patients deemed "at risk” typically, suffer from existing liver ailments, such as cirrhosis, steatosis, fibrosis, current or prior infections with hepatitis viruses, and prior treatment for HCC.
  • Early intervention can include further diagnostic assays or treatment.
  • the subject is characterized as a candidate for further diagnostic assays, such as use of ultrasound, as described supra, and/or a treatment regime. Treatments are described supra, and include surgery and ablative therapy.
  • the methods of the present invention are also useful for monitoring the efficacy or efficiency of HCC treatment that is applied to the subject after HCC has been diagnosed. Accordingly, in another aspect, the invention provides a method for determining a response of a mammalian subject having HCC tumor cells to treatment with an HCC inhibitory agent, surgical technique, or other therapeutic procedure.
  • the method can comprise: (a) determining a first level of OPN in a first biological sample taken from a mammalian subject at or prior to initiation of treatment, (b) determining a second level of OPN in a second biological sample taken from the mammalian subject taken after initiation of treatment; and (c) comparing the first and second levels of OPN, wherein a decrease in the second level of OPN measured in the second biological sample as compared to the first level of OPN measured in the first biological sample indicates a positive response to the treatment.
  • the subject is monitored multiple times during the course of HCC treatment.
  • a reduction in OPN levels corresponding to the timing of treatment indicates a positive effect in treatment to inhibit proliferation of HCC cells.
  • a reduction in OPN levels to or below the initial pre-HCC diagnostic levels indicates a substantial reduction in HCC tumor volume from the liver, and therefore, a positive response to treatment.
  • the efficacy of many cancer therapy regimes varies over time and the rates of recurrence can vary.
  • the present method can be performed to determine the efficacy or efficiency of any particular cancer therapeutic or treatment regime over time. The results of such monitoring during and after completion of therapy can help determine the prognosis for the subject and inform the ongoing and future treatment or care strategies.
  • OPN can serve as an accurate early indicator to determine the efficacy of a surgical procedure to remove an HCC tumor or tumors.
  • the levels of OPN over time after such a surgical procedure can inform whether all of the tumor or tumor mass has been removed and/or whether there is any recurrence of HCC.
  • OPN can also serve as an accurate early indicator for therapeutic response in a mammalian subject to measure the effectiveness of candidate HCC inhibitory agents, such as in a clinical trial.
  • This Example describes the discovery of osteopontin (OPN) as a biomarker for hepatocellular carcinoma (HCC) using proteomic profiling of human plasma samples.
  • OPN osteopontin
  • Plasma samples were obtained from 17 patients with HCC and 18 patients with liver cirrhosis. To reduce variability, all selected patients were male with AFP values below 20 ng/ml. Plasma was immunodepleted of human albumin, transferrin, immunoglobulin, antitrypsin and haptoglobin using the Multiple Affinity Removal Column (Agilent Technologies, Santa Clara, CA). Proteins from the immunodepleted fractions were separated using the Alliance® 2-D Bioseparations System (Waters Corporation, Milford, MA). The resulting protein fractions were further separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis. The gels were stained with colloidal Coomassie blue G-250, and each lane was cut into pieces.
  • Mass spectrometry profiling identified higher levels of osteopontin (OPN) in plasma from HCC patients, as compared to patients with liver cirrhosis.
  • OPN osteopontin
  • Extensive mass spectrometry analysis following a multi-dimensional protein separation strategy composed of two-dimensional HPLC followed by SDS-PAGE was applied to plasma samples collected from 17 patients with HCC and 18 patients with liver cirrhosis. To reduce variability, all selected patients were male with AFP values below 20 ng/ml. Osteopontin (OPN) was identified with 12 unique peptides resulting in 40.4% coverage of the full length amino acid sequence, set forth herein as SEQ ID NO:2. The sequences within the full length polypeptide sequence associated with coverage of the 12 peptide fragments are indicated as bold, underlined text in FIGURE 1A.
  • FIGURE IB is a graph illustrating OPN levels in plasma from human patients with HCC, compared to plasma of human patients with cirrhosis.
  • the mass spectrometry profile analysis identified higher levels of additional proteins in plasma from HCC patients, as compared to plasma from patients with liver cirrhosis, indicating their potential as HCC biomarkers.
  • the mass spectrometry profiling identified higher levels of lipocalin 2 (LCN2) (the full length amino acid sequence is set forth herein as SEQ ID NO: 14, encoded by the cDNA sequence set forth in SEQ ID NO: 13), nidogen 1 (NID-1) (the full length amino acid sequence is set forth herein as SEQ ID NO:20, encoded by the cDNA sequence set forth in SEQ ID NO: 19), transmembrane glycoprotein NMB (GPNMB) (the full length amino acid sequence is set forth herein as SEQ ID NO: 16, encoded by the cDNA sequence set forth in SEQ ID NO: 15), and neural cell adhesion molecule (NCAM) (the full length amino acid sequence is set forth herein as SEQ ID NO: 18, encoded by the cDNA sequence set forth in SEQ ID NO:
  • This Example describes the analysis of OPN performance as a biomarker for differentiating patients with HCC from patients with non-HCC liver disease, alone or in combination with other markers for HCC.
  • Plasma samples were collected following informed consent from patients enrolled at the University of Michigan (Cohort 1) and from patients enrolled at the Cancer Control Unit of the National Cancer Institute of Thailand, Bangkok (Cohort 2). Assays were performed at the Fred Hutchinson Cancer Research Center. The study was performed in compliance with and after approval from the respective Institutional Review Boards of all sites.
  • HCC was diagnosed according to the AASLD guidelines (Bruix et al, Hepatology 42: 1208-1236, 2005). Early stage HCC is defined as Barcelona stage 0 and A, according to AASLD guidelines and cirrhosis was defined as previously described (Marrero et al, Gastroenterology 137: 110-118, 2009).
  • HCC diagnosis was based on a clinical algorithm including imaging (ultrasonography and computerized tomography) and biochemistry (AFP and liver function enzymes testing).
  • imaging ultrasonography and computerized tomography
  • biochemistry AFP and liver function enzymes testing
  • Gender M; F (%) 82; 18 60; 40 62; 38 71; 29
  • Child-Pugh class A; B; C (%) 40; 58; 2 53; 46; 1 NA NA
  • Tumor Size (cm): ⁇ 3; 3-5;> 5 (%) 30; 55; 15 NA NA NA
  • BCLC stage 0, A; B; C (%) 13; 47; 30; 10 NA NA NA
  • Table IB Characteristics of Patients Enrolled in the Study, Cohort 2
  • Plasma levels of osteopontin (OPN) were measured using a commercial ELISA kit (R & D System, Inc., Minneapolis, MN) and 50 ⁇ of diluted (1 :100) plasma samples. Samples were added to the plates pre-coated with the first OPN-specific antibody and incubated for 2 hours at room temperature. After four washes, 200 ⁇ of the second OPN-specific antibody conjugated to horseradish peroxidase were added and incubated for 2 hours at room temperature. After four washes, 200 ⁇ of tetramethylbenzidine substrate solution were added and incubated for 30 minutes in the dark followed by the addition of 50 ⁇ of stop solution. The absorbance was measured at 450 nm with wavelength correction at 540 nm using SpectraMax® Plus 384 spectrophotometer (Molecular Devices, Sunnyvale, CA).
  • LCN2, NID-1, GPNMB, and NCAM were measured using commercially available Duoset® ELISA kits (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions.
  • ROC receiver operating characteristic
  • AUC Area under the ROC curve
  • Optimal cutoffs were calculated using the maximum sum of sensitivity and specificity as well as using the minimum distance to the top-left corner of the ROC curve.
  • the complementary property of OPN to AFP for the diagnosis of HCC was illustrated by comparing ROC curves using the Or' rule of a logistic regression with OPN and AFP in the model to that with only AFP in the model, i.e., predictor is coded as 1 if either AFP or OPN is above its respected pre-specified threshold.
  • the performance of OPN was evaluated in discriminating between HCC and cirrhosis patients in Cohort 1.
  • the Area Under the ROC Curve (AUC) for OPN (0.76, 95% CI: 0.66-0.85) was higher compared to AFP (0.71, 95% CI: 0.60-0.82) (FIGURE 3A).
  • the combination of OPN and AFP further increased the AUC (0.82, 95% CI: 0.73-0.91).
  • AUC for AFP decreased (0.64, 95% CI: 0.49-0.80) while OPN had an even higher AUC (0.80, 95% CI: 0.69-0.91) ( Figure 3B).
  • OPN OPN had a better performance than AFP with a sensitivity of 74% (95% CI: 60%-88%) compared to 53% (95% CI: 37%-69%) for AFP.
  • sensitivity increased to 85%> (95%> CI: 74%-96%) and the specificity was 63% (95%: 52%-74%).
  • the combination of OPN and AFP had a sensitivity of 71% (95% CI: 57-86) and a specificity of 86% (95% CI: 79-94).
  • the performance of OPN alone further increased with a sensitivity of 82%> (95%> CI: 66%>-98%>), while AFP performance decreased with a sensitivity of 46% (95% CI: 25%-67%) when compared to all HCC.
  • OPN again had a better performance than AFP with a sensitivity of 75% compared to 46% for AFP.
  • Cirrhosis vs HCC (AFP ⁇ 20 ng/ml)
  • ROC analysis was performed to evaluate the performance of OPN in distinguishing HCC patients from patients with cirrhosis and chronic HBV.
  • AFP performance was greater in Cohort 2 as compared to Cohort 1 , as expected since elevated levels of AFP was part of the clinical algorithm for detection of HCC.
  • the AUC for OPN (0.93, 95% CI: 0.88-0.98) was higher than for AFP (0.86, 95% CI: 0.80-0.92) (FIGURE 4B).
  • the combination of AFP and OPN had even higher AUC than OPN alone (0.96, 95% CI: 0.92-0.99).
  • OPN OPN had a better performance than AFP with a sensitivity of 93% (95% CI: 88%-98%) compared to 78% (95% CI: 70%-86%) for AFP.
  • the best cutoff for OPN determined using the minimal distance to the top-left corner in the ROC curve was 156 ng/ml in this cohort.
  • Cirrhosis/CHB vs HCC (AFP ⁇ 20 ng/ml)
  • OPN 156 74 (55-93) 96 (88-100)
  • additional markers for HCC namely LCN2, NID-1 , GPNMB, and NCAM
  • the performance of additional markers for HCC were evaluated individually and in various combinations with OPN and AFP, for the ability to discriminate between HCC and cirrhosis patients in Cohort 2.
  • the AUC values for each test are described in Table 6.
  • the combination of all six tested markers performed best for generally differentiating all cirrhosis patients from all HCC patients.
  • the combination of all six tested markers performed best for differentiating HBV-associated cirrhosis patients from HBV-associated HCC patients.
  • the combination of all six tested markers performed best for differentiating all cirrhosis patients from HCC patients with AFP values below 20 ng/ml.
  • OPN OPN-derived neurotrophic factor
  • This Example describes the discovery that elevated OPN plasma levels in at-risk patients is a biomarker for the future onset of HCC.
  • Plasma levels of OPN were assessed in 22 patients enrolled in Cohort 1 that were initially diagnosed for cirrhosis (HCC negative) but were later diagnosed with HCC upon follow-up. Cirrhosis and HCC status were determined as described above in Example 2. OPN levels were measured from plasma using ELISA and statistical analysis were performed as described above in Example 2.
  • OPN levels were investigated in patients enrolled in Cohort 1 to determine whether levels were elevated in samples collected prior to HCC diagnosis.
  • a total of 22 cirrhosis patients with prospectively collected blood specimens eventually developed HCC as determined in follow-up assessments.
  • 19 were diagnosed with early or very early stage HCC, and three were diagnosed with intermediate stage HCC (Table 7).
  • 8 of these 22 patients had AFP levels above 20 ng/ml while 19 of the 22 patients had OPN levels above 91 ng/ml, the optimal cutoff determined by the analysis of Cohort 1 samples (FIGURE 5A).
  • OPN values were below the 91ng/ml cutoff in all samples for the 22 "pre-HCC” collected more than 24 months prior to clinical HCC diagnosis (FIGURE 5D).
  • Table 7 Characteristics of patients with elevated OPN plasma levels pre -HCC dia nosis.
  • OPN Elevated levels of OPN were detected in human subjects with cirrhosis, but had no clinically detectable HCC, at time points within 24 months before the patients were eventually diagnosed with HCC in follow-up exams. Therefore, the utility of OPN as a biomarker for development HCC prior to development of clinically detectable HCC was demonstrated.
  • This Example describes the discovery that levels of osteopontin (OPN) splice variant isoforms are increased in patients with HCC.
  • OPN osteopontin
  • human OPN has two additional isoforms reflecting splice variants, indicated as isoforms B, and C.
  • the polypeptide sequence of isoform B is set forth herein as SEQ ID NO:4, and is encoded by the cDNA sequence set forth in SEQ ID NO:3.
  • the polypeptide sequence of isoform C is set forth herein as SEQ ID NO:6, and is encoded by the cDNA sequence set forth in SEQ ID NO:5.
  • Different functions have been attributed to these different isoforms. The presence and abundance of the splice variant isoforms in patients with HCC was investigated.
  • Mass spectrometry profiling was performed on plasma samples from eight human patients with liver cirrhosis and 12 human patients with diagnosed HCC, according to the general protocol described in Example 1.
  • mRNA levels for all three OPN isoforms in human HCC tumors were assayed by quantitative PCR.
  • HCC tumors were obtained from six human patients by standard biopsy procedures. Total RNA was isolated from the tumor tissue by standard techniques. The fold change in mRNA levels was assayed employing primers unique to each isoform (A, B, and C) mRNA (corresponding cDNA sequences set forth in SEQ ID NOS: l, 3, and 5, respectively) under standard cycling conditions.
  • the forward and reverse primers used to specifically amplify and detect mRNA encoding OPN isoform A are set forth herein in SEQ ID NOS:7 and 8, respectively.
  • the forward and reverse primers used to specifically amplify and detect mRNA encoding OPN isoform B are set forth herein in SEQ ID NOS:9 and 10, respectively.
  • the forward and reverse primers used to specifically amplify and detect mRNA encoding OPN isoform A are set forth herein in SEQ ID NOS: l l and 12, respectively.
  • the fold change of mRNA levels was expressed relative to levels detected in a pool of normal liver tissue.
  • FIGURE 6A is a graph illustrating OPN isoform B levels in plasma from patients with HCC, compared to plasma from human patients with cirrhosis. As illustrated, multiple peptide fragments specific for OPN isoform B (SEQ ID NO:4) were detected in plasma from multiple HCC patients, but were not detected in plasma from cirrhosis patients.
  • FIGURE 6B is a graph illustrating the fold change in mRNA for all three OPN splice variant isoforms in human HCC tumors compared to levels detected in a pool of normal liver tissue. As illustrated, mRNA levels increased for all three isoforms in HCC tumors. The median increases for the OPN isoforms were between approximately 20 and 40 fold.
  • Pten null mice contain liver specific deletion of the phosphoinositide 3 -kinase
  • mice develop NASH, fibrosis and HCC later in life, making them a highly relevant model for the human HCC disease.
  • Pten mouse model for HCC was studied for biomarkers predictive of the onset of HCC.
  • Extensive mass spectrometry-based profiling was performed to characterize liver and plasma proteome changes associated with fibrosis, NASH or HCC, in the Pten null and other mouse models.
  • Proteomic screening was performed in a similar manner as described in Example 1 in the context of screening human serum. Overall, a total of 8,892 liver proteins and 3,694 plasma proteins were identified and quantified. As part this analysis, HCC-associated changes in extracellular matrix proteins (ECM) and their receptors was observed, contributing to the ongoing efforts to use ECM composition as diagnostic markers and therapeutic targets.
  • ECM extracellular matrix proteins
  • the proteomic study also identified osteopontin (OPN) and lipocalin 2 (LCN2) as biomarkers for the development of HCC tumors.
  • OPN osteopontin
  • LN2 lipocalin 2
  • the amino acid sequence for mouse OPN is set forth herein as SEQ ID NO:22, and is encoded by the nucleic acid set forth in SEQ ID NO:21.
  • the amino acid sequence for mouse LCN2 is set forth herein as SEQ ID NO:24, and is encoded by the nucleic acid set forth in SEQ ID NO:23.
  • FIGURES 7A-D graphically illustrate the OPN and LCN2 protein abundance in the tissue and plasma of mice.
  • OPN protein levels are greatly elevated in HCC tumor tissue compared to liver tissue of control mice and mice with NASH.
  • OPN protein levels are elevated in the plasma of Pten mice, and increase over time, with the highest levels in mice with HCC.
  • LCN2 protein levels are elevated in HCC tumor tissue compared to liver tissue of control mice and mice with NASH.
  • OPN and LCN2 are upregulated in mice with HCC, and are detectable in both the liver tissue and plasma. Furthermore, the detectable levels of OPN and LCN2 positively correlate with the progressive increase of tumor size and development. Therefore, the utility of OPN and LCN2 as biomarkers for the development and relative progression of HCC is demonstrated.
  • This example describes the development of a mass-spectrometry based assay to detect and quantify OPN isoform tryptic peptides in the femtomole concentration range using stable heavy-labeled isotopic standards as reference peptides.
  • a stock solution of three custom synthetic peptides and their stable-labeled isotopically “heavy" analogs was prepared by mixing all of the peptides in 95:5:0.1 % water :acetonitrile: formic acid (v:v:v) diluent to a final concentration of 100 fmol/ ⁇ .
  • This stock solution was analyzed on a mass spectrometer (Thermo Scientific TSQ Vantage Quadrupole mass spectrometer, Thermo- Fisher Scientific, Waltham, MA) in the full-scan mode to obtain MS/MS fragmentation information.
  • the mass spectrometer used the Xcalibur control software (ver 2.1) with Thermo EZ-Tune (ver 2.3 SP1) and was coupled to a 2 dimensional HPLC system (Eksigent NanoLC 2-deminsional HPLC system, Dublin, CA). A 5 ⁇ aliquot was loaded onto a 15 cm 75 ⁇ i.d. PicoFrit capillary column (New Objective, Woburn, MA) packed with C 18 reverse-phase resin and eluted with a linear acetonitrile/water gradient (2 - 40 % "B" in 10 minutes, A: 0.1 % formic acid, B: acetonitrile/01 % formic acid) at 400 nl/minute.
  • the solvents were of LC-MS grade from Burdick and Jackson (Honeywell, Morristown, NJ). MS and MS/MS spectra were collected in Q3 mode with scan times of 0.5 and 1.0 seconds respectively; the MS/MS energy ramp was set at 30 volts with a 0.035 volt per m/z gradient.
  • the capillary temperature was 275 °C and the spray voltage was 1800.
  • the four most-intense b- or y- ions were chosen for use as transitions.
  • the software exported a range of collisional energy voltages which spanned ⁇ 5 volts from a theoretically calculated optimal value as to give a range to voltages to evaluate. 5 ⁇ aliquots of the 100 fmol/ ⁇ heavy reference/light peptide mixture were run on the same 15 cm capillary column for the CE optimization runs. Data were then imported back into Skyline for evaluation; the CE voltage which yielded the highest intensity peak for each peptide was determined to be the optimal value.
  • a depleted mouse serum sample was then analyzed on the mass spectrometer with the optimized method.
  • a stock solution of all "heavy" reference peptides was prepared by mixing all in the 95:5 :0.1% water :acetonitrile: formic acid diluent to a final concentration of 250 fmol/ ⁇ .
  • Two microliters of the "heavy" stock were mixed with 8 ⁇ of 0.25 ⁇ g/ ⁇ l serum sample to yield final concentrations of 0.2 ⁇ g/ ⁇ l of total peptides with 50 fmol/ ⁇ of each "heavy" reference peptide.
  • a 5 ⁇ aliquot of the mixture was run in duplicate and the raw data were imported into Skyline for evaluation. Each peak was visually inspected to 1) verify a peak was identified 2) ensure that the peak was correctly identified at the expected elution time and 3) integration baselines were adjusted to ensure all transitions were used to contribute to the peak's total integrated area.
  • the compiled data from Skyline was exported to a CSV formatted file using Skyline's built-in "export report” function.
  • the total integrated area (summed from the area contributions of each transition) each peak was verified and then summarized to allow calculation of the endogenous peptide concentration from the ratio of "heavy" reference peptide area vs. "light” endogenous peptide area.
  • Table 8 summarizes the transitions and their optimized CE's used in the SRM method. Concentrations of the endogenous peptides were calculated using the rations of the area observed for the "light and "heavy" standard peptides as follows:
  • Endogenous Concentration (total endogenous peptide loaded/injection volume) x (dilution factor)
  • Total endogenous peptide loaded Integrated Light Area x (250 thiol "heavy” standard/Integrated "heavy” area
  • Table 9 shows the calculated concentrations of endogenous peptides.
  • HepG2 supematant-01 0.5 0.2 0.0
  • HepG2 supematant-02 0.4 0.2 0.0
  • a SRM-based mass spectrometry assay was developed for quantification of endogenous peptides in serum samples.
  • CE collisional energy
  • An optimized SRM acquisition method was set-up and endogenous-containing peptide mixtures spiked with "heavy" reference standards was analyzed. The acquired raw data was loaded into Skyline and peak areas were integrated within the software. The results were exported into an excel spreadsheet for reporting purposes and a calculation of the endogenous peptide concentration was made by ratio of the area of "heavy" reference standard vs. the area of the endogenous peptide(s).
  • the measured endogenous peptide concentrations were found to be within the ranges of 0 to 10.2 fmol/ul, a range that is most likely below the limit of quantification for each assay.
  • the peptide sample for CS32 was rerun due to low signal observed during the first run.
  • This example describes the assay of OPN and LCN2 in Korean serum samples.
  • the serum levels of OPN and LCN2 were measured by commercial ELISA in a Korean cohort that includes 160 samples, 105 samples from cirrhotic patients (62M/ 43F, median age 52, range 33-72) and 55 sample from HCC patients (42M/13F, median age 58, range 35-84).
  • OPN shown in FIG 10A
  • LCN2 shown in FIG 10B
  • AFP alpha-fetoprotein
  • DCP des-y-carboxyprothrombin
  • FIGURE 11 A-D the area under the curve (AUC) for each marker alone was: 0.832 for AFP (FIGURE 11A), 0.864 for DCP (FIGURE 1 IB), 0.862 for OPN (FIGURE 11C) and 0.715 for LCN2 (FIGURE 11D).
  • the combination of two markers gave the following AUC values: 0.885 for AFP+DCP; 0.936 for AFP+OPN; 0.876 for AFP+LCN2; 0.924 for DCP+OPN; 0.901 for DCP+LCN2; and 0.873 for OPN+LCN2.
  • the AUC values for three markers combined were: 0.946 for AFP+OPN+LCN2; 0.934 for DCP+OPN+LCN2; 0.935 for AFP+DCP+OPN; and 0.92 for

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Abstract

La présente invention concerne la détermination de la présence d'un carcinome hépatocellulaire (CHC) chez un sujet mammifère, ou l'évaluation du risque de développer un carcinome hépatocellulaire (CHC) chez un sujet mammifère, avant le développement d'un CHC cliniquement détectable. Lesdits procédés comprennent la détermination du niveau d'ostéopontine (OPN) dans un échantillon biologique prélevé sur un sujet, et la comparaison dudit niveau à un étalon de référence ou une valeur de seuil. Un niveau élevé d'OPN comparé à l'étalon de référence ou à la valeur de seuil indique la présence de HCC chez un sujet, ou un risque accru pour le sujet de développer un CHC cliniquement détectable.
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CN104459161A (zh) * 2014-12-30 2015-03-25 霍普金斯医药研究院（北京）有限责任公司 一种甲胎蛋白胶体金检测试剂盒及其使用方法
RU2723891C1 (ru) * 2019-09-23 2020-06-18 Федеральное государственное бюджетное образовательное учреждение высшего образования Иркутский государственный медицинский университет Министерства здравоохранения Российской Федерации Способ определения риска развития гепатоцеллюлярной карциномы у больных хроническим гепатитом с
WO2022187228A1 (fr) * 2021-03-01 2022-09-09 Board Of Regents, The University Of Texas System Procédés de détection de biomarqueurs de fibrose hépatique ou de carcinome hépatocellulaire avancés dans un échantillon

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104459161A (zh) * 2014-12-30 2015-03-25 霍普金斯医药研究院（北京）有限责任公司 一种甲胎蛋白胶体金检测试剂盒及其使用方法
RU2723891C1 (ru) * 2019-09-23 2020-06-18 Федеральное государственное бюджетное образовательное учреждение высшего образования Иркутский государственный медицинский университет Министерства здравоохранения Российской Федерации Способ определения риска развития гепатоцеллюлярной карциномы у больных хроническим гепатитом с
WO2022187228A1 (fr) * 2021-03-01 2022-09-09 Board Of Regents, The University Of Texas System Procédés de détection de biomarqueurs de fibrose hépatique ou de carcinome hépatocellulaire avancés dans un échantillon

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