Classifications

• • 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
  • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
  • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
  • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
• • 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
  • G01N33/57423—Specifically defined cancers of lung
• • 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
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/60—Complex ways of combining multiple protein biomarkers for diagnosis

Definitions

• the invention relates to materials and methods for diagnosing tumor types, and assessing patient prognosis.
• the invention concerns the determination of marker proteins which enable primary liver tumors to be identified and classified.
• the liver is a complex organ capable of regeneration after damage. It is highly structured with a number of specialised cells required to form amongst other features, the bile ducts and liver parenchyma. The most common cell type is the hepatocyte that forms the bulk of the liver parenchyma. Cholangiocytes are a much less common cell type forming the bile ducts of the intrahepatic biliary tree.
• HCC Hepatocellular carcinoma
• CC cholangiocarcinoma
• CC can arise from any portion of the intrahepatic biliary tree, and is classified into peripheral and hilar/perihilar based on the predominant location, probable different biological characteristics, and pathogenesis. This classification is supported by an association with risk factors such as viral hepatitis or alcoholic liver disease in peripheral CC.
• risk factors such as viral hepatitis or alcoholic liver disease in peripheral CC.
• PSC primary sclerosing cholangitis
• Some primary carcinomas show a mixed phenotype, with areas of hepatocellular differentiation alternating with areas of cholangiocellular differentiation.
• An origin from hepatic progenitor cells has been proposed for these tumors, on the broader basis of the cancer stem cell theory that all primary liver tumors and in particular the epithelial ones may be part of a phenotypic spectrum with "pure" HCC and CC at either end, and mixed cancers somewhere in the middle [4, 5] .
• TACE transarterial chemoembolization
• a potential explanation for this observation is that TACE provides selection pressure in favor of a minor progenitor cell population that is resistant to TACE and capable of multipotent differentiation including biliary lineage.
• TACE transarterial chemoembolization
• cholangiocellular/progenitor cell components were identified by single or double immunostainings or gene expression analysis (RT-PCR) from
• markers are required to help better define the details of the phenotype and pathogenesis of the different HCC/CC components of post-TACE tumors, their similarities to their normal and typical malignant counterparts, and aid in diagnosis, prognosis and potentially identify new selective therapeutic targets and predictive markers.
• LC-MS/MS Liquid chromatography - mass spectrometry
• Microdissection to enrich tumor cell populations from heterogenous tissue sections Microdissection to enrich tumor cell populations from heterogenous tissue sections .
• Standard liver histology and immunohistochemistry for tumor marker marker proteins have provided some means of differentiating between HCC and CC but are prone to inter-operator variability and lack of sensitivity.
• a need for more informative markers for characterising liver tumors in terms of the predominant cellular type - hepatocytes or cholangiocytes - and potentially incorporating molecular markers of drug responsiveness.
• biomarkers of tumor cell lineage can provide an aid to earlier diagnosis, prognostic monitoring of disease, optimised treatment selection and may potentially identify new selective therapeutic targets for future drug development .
• the present invention therefore, provides for novel biomarkers for use in the classification of primary liver tumors and particularly distinction between hepatocellular carcinoma and cholangiocellular carcinoma.
• the present invention provides for a method of determining the cellular phenotype of a liver tissue sample said method comprising
• step (2) determining expression levels of a plurality of marker proteins in said sample, wherein said plurality of marker proteins are selected from a biomarker panel as represented by any one of Table 1A, Tables 2 to 11; optionally, repeating step (2) with a different plurality of marker proteins selected from a biomarker panel as represented by any one of Table 1A, Tables 2 to 11;
• a method of identifying the cellular phenotype of a liver cell comprising
• the plurality of marker proteins are selected from a biomarker panel as represented by any one of Table 1A, Tables 2 to 10 or Table 11.
• the cellular phenotype is selected from normal liver epithelium cells (hepatocytes ) , normal biliary epithelium cells (cholangiocytes ) , hepatocellular carcinoma cells, peripheral cholangiocellular carcinoma cells or hilar cholangiocellular carcinoma cells.
• the methods further comprise comparing said expression levels with a second reference set of expression levels representing a second cellular phenotype.
• the liver cell is a liver tumor cell.
• the biomarker panel is represented by Table 5 and/or Table 7 and the cellular phenotype is selected from hepatocellular carcinoma cells and cholangiocellular carcinoma cells, preferably the plurality of marker proteins is selected from part A of Table 5.
• the liver tumor cell is obtained from a liver tumor biopsy sample, preferably obtained from a patient having previously been treated with transarterial chemoembolization.
• the plurality of marker proteins are selected from Table 7, preferably the plurality of marker proteins are selected from Table 7 part A.
• the step of determining the expression levels of a plurality of marker proteins comprises (a) contacting the liver cell or the liver tissue sample with a plurality of binding members, wherein each binding member selectively binds to one of said plurality of marker proteins or nucleic acid sequences encoding said marker proteins;
• the specific binding member is an antibody or antibody fragment which selectively binds to one of said plurality of marker proteins or a nucleic acid sequence which selectively binds to nucleic acid encoding one of said plurality of marker proteins .
• the specific binding member is an aptamer or the binding member is immobilised on a solid support.
• the step of determining expression levels of a plurality of marker proteins is performed by mass spectrometry or by Selected Reaction Monitoring using one or more transitions for protein derived peptides; and comparing the peptide levels in the liver cell or the liver tissue sample under test with peptide levels previously determined to represent a cellular phenotype .
• comparing the peptide levels includes determining the amount of protein derived peptides from the liver cell or the liver tissue sample with known amounts of corresponding synthetic peptides, wherein the synthetic peptides are identical in sequence to the peptides obtained from the liver cell or the liver tissue sample except for a label. More preferably, the label is a tag of a different mass or a heavy isotope.
• the present invention provides for a method for the diagnosis or prognostic monitoring of a liver tumor in an individual, said method comprising
• the present invention provides for a method for determining a treatment regimen for an individual having a liver tumor, said method comprising
• the liver tumor cell is from a liver tumor biopsy.
• the biomarker panel is represented by Table 5, preferably by Part A of Table 5.
• the individual had previously been treated with transarterial chemoembolization .
• the biomarker panel is represented by Table 7, more preferably by Part A of Table 7.
• the present invention provides for a method of diagnosing liver cancer in an individual comprising detecting one or more marker proteins or fragments thereof selected from Table 1A, Tables 2 to 11 in a blood, tissue, saliva or urine sample obtained from said individual.
• said one or more protein markers or fragments thereof are detecting using a specific binding member, more preferably said binding member is an antibody specific for said one or marker protein .
• the plurality of marker proteins are selected from any one of Collagen alpha 1 (XVIII) chain, Plastin-3, AKR1B10, Fibronectin, Beta 3 tubulin, Asporin, 14-3-3 protein eta or Dihydropyrimidinase-related protein 3 or combinations thereof, preferably the plurality of marker proteins comprises AKR1B10 and/or Beta 3 tubulin.
• the present invention provides for e use of one or more marker proteins selected from Table 1A, Tables 2 to 11 as a agnostic marker for liver cancer .
• the present invention provides for a method for diagnosing recurrent or primary liver tumor in a subject, the method comprising determining the presence or absence of one or more marker proteins selected from the group consisting of Collagen alpha 1 (XVIII) chain, Plastin-3, AKRIBIO, Fibronectin, Beta 3 tubulin, Asporin, 14-3-3 protein eta, and Dihydropyrimidinase-related protein 3 in a sample.
• the liver tumor is selected from the group consisting of hepatocellular carcinoma, peripheral cholangiocellular carcinoma or hilar cholangiocellular carcinoma cells.
• the marker protein is Beta 3 tubulin and/or AKRIBIO, preferably Beta 3 tubulin.
• the sample is selected from any one of blood, plasma, serum, liver tissue, liver cells or combinations thereof, preferably the sample is liver tissue, optionally formalin-fixed paraffin-embedded liver tissue section.
• the determining the presence or absence of one or more marker proteins in the sample is performed by either Immunohistochemistry (IHC) or mass spectrometry.
• IHC Immunohistochemistry
• the invention provides for a kit for diagnosing recurrent or primary liver tumor in a subject, the kit comprising reagents for determining the presence or absence of one or more marker proteins selected from the group consisting of Collagen alpha 1 (XVIII) chain, Plastin-3, AKRIBIO, Fibronectin, Beta 3 tubulin, Asporin, 14-3-3 protein eta, and Dihydropyrimidinase-related protein 3 in a sample.
• the liver tumor is selected from the group consisting of hepatocellular carcinoma, peripheral cholangiocellular carcinoma or hilar cholangiocellular carcinoma cells.
• the marker protein is Beta 3 tubulin and/or AKRIBIO, preferably Beta 3 tubulin.
• the kit comprises reagents suitable for preparing the sample, wherein the sample is selected from any one of blood, plasma, serum, liver tissue, liver cells or combinations thereof.
• the sample is liver tissue and the kit comprises reagents suitable for preparing liver tissue, optionally for preparing formalin-fixed paraffin-embedded liver tissue sections.
• the determining the presence or absence of of one or more marker proteins in the sample is performed by either Immunohistochemistry .
• the present invention provides for a kit for use in determining the cellular phenotype of a liver cell, said kit allowing the user to determine the presence or level of expression of a plurality of analytes selected from proteins or fragments thereof provided in biomarker panels as represented by any one of Table 1A, Tables 2 to 11, a plurality of antibodies against said marker proteins and a plurality of nucleic acid molecules encoding said marker proteins or fragments thereof, in a cell under test; the kit comprising
• the present invention also provides for a kit for use in determining the cellular phenotype of a liver cell in vitro, said kit allowing the user to determine the presence or level of expression of a plurality of proteins or fragments thereof provided in biomarker panels represented by Table 1A, Tables 2 to 11, in a cell under test; the kit comprising
• the present invention provides for a kit for the diagnosis, prognostic monitoring of a liver tumor in an individual or for determining a treatment regimen for an individual having a liver tumor, the kit comprising
• each binding member selectively binds to a protein selected from a biomarker panel as represented by any one of Table 1A, Tables 2 to 11; or a nucleic acid encoding the protein or fragment thereof;
• a developing agent comprising a label
• one or more components selected from washing solutions, diluents and buffers .
• the biomarker panel is represented by Table 5 or by Part A of Table 4 or by by Table 7 or Part A of Table 7.
• the present invention provides for a plurality of synthetic peptides each having a sequence identical to a fragment of one of a plurality of proteins selected from a biomarker panel selected from any one of Table 1A, Tables 2 to 11, said fragment resulting from digestion of the protein by trypsin, ArgC, AspN or Lys-C digestion, wherein one or more of the plurality of synthetic peptides comprises a label, optionally for the use in Selective Reaction Monitoring
• the label is a heavy isotope.
• the present invention also provides for a liver cellular classification system comprising a liver cellular classification apparatus and an information communication terminal apparatus, said liver cellular classification apparatus including a control component and a memory component, said apparatuses being communicatively connected to each other via a network;
• the information communication terminal apparatus includes
• liver cellular classification apparatus includes
• the memory unit contains data of a plurality of proteins selected from Table 5 or Table 11 and wherein the classification is between Hepatocellular carcinoma and peripheral cholangiocarcinoma; alternatively the memory unit contains data of a plurality of proteins selected from Table 7 or Table 11 and wherein the classification is between Hepatocellular carcinoma and cholangiocarcinoma in post-TACE liver tumors.
• the liver cellular classification system is connected to an apparatus for determining the protein expression levels in a liver tissue sample, preferably the apparatus can process multiple samples using liquid chromatography-mass spectrometry (LC-MS/MS) .
• LC-MS/MS liquid chromatography-mass spectrometry
• a liver tissue cellular classification program that makes an information processing apparatus including a control component and a memory component execute a method of determining and/or classifying the liver tissue of a subject, the method comprising :
• a classifying step for classifying the liver tissue cells of said subject, based on the comparison calculated at the comparing step; and wherein said tissue is classified into phenotypes including normal (hepatocytes , cholangiocytes ) , hepatocellular carcinoma, truly mixed hepatocholangiocellular carcinoma (pre or post TACE therapy) , peripheral cholangiocarcinoma, Hilar cholangiocarcinoma (with or without primary sclerosing cholangitis), or metastatic colo-rectal carcinoma.
• the liver tissue cellular classification program of claim 48 recorded thereon.
• FIG. 1 Overall workflow. Overall data analysis workflow; Spectrum files (0), Spectrum Selector (1), Sequest (2), Percolator (3), Mascot (4), Event Detector (5) , Precursors Ions Area Detector (6) , Peptide Area Quantified (7) , Peptide Identification at 1%FDR + spectral counting (8) , Peptide Matrix with Area under the Curve (AUC) (9), Peptide data matrix with spectral count information (10), Statistical validation (11; 12) and final list (13) .
• AUC Area under the Curve
• Figure 2 Venn-Diagram. The diagram shows comparison of the two quantitation methods (left: spectral count; right: Area under the Curve) for marker proteins with unique and shared peptides . The numbers shown the marker proteins found to be significantly modulated in each quantification method and those common to both, across all comparisons made in this study.
• PCA Principal component analysis
• FIG. 4 Validation of protein up-regulation through Volcano plots, (a) Volcano plots for AKR1B10 (upper left panel, normal liver parenchyma (1) vs. HCC (2); upper right panel, normal liver parenchyma (1) vs. normal bile duct (9) ) and Tubulin-beta 3 chain (lower left panel, normal liver parenchyma (1) vs. peripheral CC (5); lower right panel, peripheral CC (5) vs. normal bile duct (9) .
• AKR1B10 upper left panel, normal liver parenchyma (1) vs. HCC (2); upper right panel, normal liver parenchyma (1) vs. normal bile duct (9)
• Tubulin-beta 3 chain lower left panel, normal liver parenchyma (1) vs. peripheral CC (5); lower right panel, peripheral CC (5) vs. normal bile duct (9) .
• FIG. 1 tissue type 1 (normal liver); (2) tissue type 2 (HCC); (3) tissue type 9 (Normal bile duct) and (4) tissue type 5 (peripheral CC) .
• AKR1B10 is diffusely expressed in HCC, while its expression is only patchy or weak in normal liver parenchyma and peripheral CC.
• AKR1B10 is also diffusely positive in normal bile duct.
• Immunostaining for Tubulin-beta 3 chain on normal liver, HCC, normal bile duct, and peripheral CC The expression of Tubulin-beta 3 chain appears to be specific for peripheral CC .
• Table 11 shows a list of marker proteins (467) with both unique and shared peptide sequences .
• pluripotenty of marker proteins means at least two marker proteins as disclosed herein.
• marker protein includes all biologically relevant forms of the protein identified, including post-translational modifications.
• the marker protein can be present in a glycosylated, phosphorylated, multimeric, fragmented or precursor form.
• a marker protein fragment may be naturally occurring or, for example, enzymatically generated and the biologically active function of the full marker protein. Fragments will typically be at least about 10 amino acids, usually at least about 50 amino acids in length, and can be as long as 300 amino acids in length or longer.
• cellular phenotype refers to the characteristics or traits of a cell or group of cells.
• Cellular phenotype refers to the cells anatomical location, morphology, development, biochemical or physiological properties, behaviour, and products of biochemistry/behaviour. Cellular phenotype results from the expression of cell genes as well as the influence of environmental factors and the interactions between the two.
• liver tissue sample include, but is not limited to, a specimen of liver tissue removed by resection or core needle biopsy.
• expression level refers to the relative amount of protein in a liver tissue sample, for example as determined by LC-MS/MS label free quantification approaches such as area under the curve and spectral counting.
• comparing means measuring the relative amount of a protein or proteins in a sample relative to other samples (for example protein amounts stored in our database) .
• reference set refers to the samples (for example in our database) used as classifiers (e.g. classic examples or HCC, or CC) . These classifiers can be used to help diagnosis of non-classic specimens from new cases.
• reference level refers to a pre-determined level, which may, for example be provided in the form of an accessible data record from a public database.
• antibody includes polyclonal antiserum, monoclonal antibodies, fragments of antibodies such as single chain and Fab fragments, and genetically engineered antibodies.
• the antibodies may be chimeric or of a single species.
• marker protein and “biomarker”, which are used interchangeably herein, include all biologically relevant forms of the protein identified, including post-translational modifications.
• the marker protein can be present in a glycosylated, phosphorylated, multimeric or precursor form .
• control refers to a cultured cell line, primary culture of cells taken from a human or animal subject, or biopsy material taken from a human or animal subject that is free of HCC or CC .
• antibody array or "antibody microarray” means an array of unique addressable elements on a continuous solid surface whereby at each unique addressable element an antibody with defined specificity for an antigen is immobilised in a manner allowing its subsequent capture of the target antigen and subsequent detection of the extent of such binding.
• Each unique addressable element is spaced from all other unique addressable elements on the solid surface so that the binding and detection of specific antigens does not interfere with any adjacent such unique addressable element.
• bead suspension array means an aqueous suspension of one or more identifiably distinct particles whereby each particle contains coding features relating to its size and colour or fluorescent signature and to which all of the beads of a particular combination of such coding features is coated with an antibody with a defined specificity for an antigen in a manner allowing its subsequent capture of the target antigen and subsequent detection of the extent of such binding. Examples of such arrays can be found at
• SRM selected reaction monitoring
• MRM mass spectrometry assay whereby precursor ions of known mass-to-charge ratio representing known biomarkers are preferentially targeted for analysis by tandem mass spectrometry in an ion trap or triple quadrupole mass spectrometer. During the analysis the parent ion is fragmented and the number of daughter ions of a second predefined mass-to-charge ratio is counted. Typically, an equivalent precursor ion bearing a predefined number of stable isotope substitutions but otherwise chemically identical to the target ion is included in the method to act as a quantitative internal standard.
• differential expression refers to at least one recognisable difference in protein expression. It may be a quantitatively measurable, semi-quantitatively estimable or qualitatively detectable difference in tissue protein expression.
• a differentially expressed protein may be strongly expressed in tissue in one cellular phenotype (e.g. HCC) and less strongly expressed or not expressed at all in another cellular phenotype (e.g. CC) . Further, expression may be regarded as differential if the protein undergoes any recognisable change such as cleavage or post-translational modification between two cellular phenotypes under comparison.
• isolated means throughout this specification, that marker protein, antibody or polynucleotide, as the case may be, exists physical milieu distinct from that in which it may occur in nature.
• subject includes any human or non-human animal.
• non-human animal includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc.
• treat includes therapeutic treatments, prophylactic treatments and applications in which one reduces the risk that a subjectwill develop a disorder or other risk factor. Treatment does not require the complete curing of a disorder and encompasses the reduction of the symptoms or underlying risk factors .
• LMD laser microdissection
• TACE trans-arterial chemo-embolization
• HCC hepatocellular carcinoma
• CC cholangiocellular carcinoma
• PSC primary sclerosing cholangitis
• FFPE Formalin Fixed Paraffin embedded
• AUC area under the curve
• PSM peptide spectrum match.
• the inventors have identified marker proteins that demonstrate statistically significant differences in protein expression levels between different cellular phenotypes of liver cells, including liver tumor cells .
• the inventors have determined marker proteins having different expression levels between components (HCC and CC) of post-TACE HCC, Often cases diagnosed with HCC are then treated with transarterial chemoembolization (TACE) , however tumors generally come back, but no longer show the classic HCC phenotype, having some regions that look classic HCC, some that look classic CC, and some which are undefinable.
• TACE transarterial chemoembolization
• the present invention allows for the identification of marker proteins more specific for HCC than CC, or vice versa, in a patient that has already undergone TACE.
• the inventors have further explored their similarities or dissimilarities compared to their normal and typical malignant counterparts .
• the inventors also found significant differences in other tissue type comparisons. These differentially expressed marker proteins provide useful biomarkers to help diagnosing tumor types, assessing patient prognosis and determining appropriate treatment regimens .
• marker protein sets or biomarker panels
• biomarker panels specific to the hepatocellular and cholangiocellular phenotype of post-TACE mixed tumors, and their similarity to their normal and typical neoplastic counterparts confirms that the differentiation process is truly divergent, despite a probable origin from a common progenitor.
• marker proteins differentially expressed between normal and neoplastic hepatocytes and biliary epithelial cells, as they provide new markers of malignant transformation or tumor differentiation; and between HCC and peripheral CC, which often overlap in both clinical presentation, and appearance on imaging and histology (22, 23) .
• the present invention provides herein marker proteins which are differentially expressed between two cell types tested and allow a particular cellular phenotype to be determined.
• Table 1A shows the preferred marker proteins (including their synonyms) according to the invention, namely Beta 3 tubulin, AKR1B10, Collagen alpha 1 (XVIII) chain, Plastin-3, Fibronectin, Asporin, 14-3-3 protein eta and
• Pre-TACE pre-trans-arterial chemo-embolization
• HCC hepatocellular carcinoma
• CC cholangiocellular carcinoma
• PSC primary sclerosing cholangitis
• Table IB indicates the numbers of proteins that showed statistically significant differential expression levels between two types of liver tissues (p-value ⁇ 0.05 and Log2 [fold change] ⁇ 2 or ⁇ -2 ) using shared and unique peptides These numbers illustrate the number of differentially modulated proteins that were common to both area under the curve and spectral counting datasets per tissue type comparison (467 proteins common to both) .
• Table IB Number of proteins showing statistically significant differential expression between types of liver tissue.
• PSC metastatic colorectal carcinoma.
• the marker proteins indicated in the Tables 2 to 10 allow the following cell types to be distinguished : -
• Table 2 Normal hepatocytes from HCC .
• Table 3 Peripheral cholangiocarcinoma from normal bile duct.
• Table 4 Hilar cholangiocarcinoma from normal bile duct.
• Table 5 Hepatocellular carcinoma from peripheral cholangiocarcinoma.
• Table 6 Hepatocytes from cholangiocytes .
• Table 7 Hepatocellular carcinoma and cholangiocarcinoma in post-TACE liver tumors .
• Table 8 Peripheral cholangiocarcinoma from metastatic colorectal cancer.
• Table 9 Hilar cholangiocarcinoma from hilar cholangiocarcinoma with primary sclerosing cholangitis.
• Table 10 Hilar cholangiocarcinoma from metastatic colorectal cancer.
• Q-values (adjusted p values) provide a more stringent measure of statistical significance than p-values and were computed using a direct False Discovery Rate approach. Individual Q-values are not shown here but all marker proteins with q-values ⁇ 0.05 are listed in section A of each tables 2 to 10, while all marker proteins with p-values ⁇ 0.05 are displayed in section B of each table 2 to 10.
• Protein expression levels for marker proteins shown in Tables 2 to 10 were determined using label free LC-MS/MS quantification based on spectral counting ( shared and unique peptides ) which is well known in the art . All marker proteins showing statistically significant differences in mean spectral counts between two tissue types are display in Tables 2-10. We have also used an alternate method of data analysis based on the area under the curve (AUC) of the MSI peak of the three most intense peptides for each protein. All marker proteins in Table 11 ( Figure 7) marker proteins (467 marker proteins) were found to be significantly regulated in at least one of the tissue comparisons that were common to both quantification methods (spectral counting and AUC of both shared and unique peptides) .
• the Table contains tissue type comparison (Tissue type number versus tissue type number) , uniprot ID, and protein names along with P-values, t-scores and log 2 Fold-change values for both quantitative methods.
• Table 2 provides protein markers for use in distinguishing normal hepatocytes from hepatocellular carcinoma cells (HCC) .
• *Bold type indicates increased relative expression in hepatocellular carcinoma compared to normal hepatocytes.
• Table 2 provides information as to whether the marker proteins are relatively over-expressed (identified in bold) or under-expressed in HCC versus normal hepatocytes. Accordingly, by determining the presence, absence or change in expression levels of a plurality of these marker proteins and comparing these changes with a reference of known expression levels, one is able to determine whether the cells under test are HCC or normal hepatocytes.
• the plurality of marker proteins may be selected from Table 2 as a whole, or preferably from Part A which lists those marker proteins showing a higher statistically significant difference between the two cell types.
• Table 3 provides information as to whether the marker proteins are relatively over-expressed (identified in bold) or under-expressed in peripheral cholangiocarcinoma versus normal cholangiocytes . Accordingly, by determining the presence, absence or change in expression levels of a plurality of these marker proteins and comparing these changes with a reference of known expression levels, one is able to determine whether the cells under test are peripheral cholangiocarcinoma or normal cholangiocytes .
• P13797 Plastin-3 / Name PLS3 5.39E-03 1 .76 1 .76 8.38 8.38
• Table 4 provides information as to whether the marker proteins are relatively over-expressed (identified in bold) or under-expressed in hilar
• cholangiocarcinoma versus normal cholangiocytes . Accordingly, by determining the presence, absence or change in expression levels of a plurality of these marker proteins and comparing these changes with a reference of known expression levels, one is able to determine whether the cells under test are hilar cholangiocarcinoma or normal cholangiocytes.
• P60842 Eukaryotic initiation factor 4A-I / 8.54E-03 -1 .62 1.62 -5.95 5.95
• Table 5 provides information as to whether the marker proteins are relatively over-expressed (identified in bold) or under-expressed in peripheral carcinoma versus hepatocellular carcinoma. Accordingly, by determining the presence, absence or change in expression levels of a plurality of these marker proteins and comparing these changes with a reference of known expression levels, one is able to determine whether the cells under test are hepatocellular carcinoma or peripheral carcinoma.
• P80404 4-aminobutyrate 1 .16E-03 2.83 2.83 24 24 aminotransferase

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