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
  • G01N33/57419—Specifically defined cancers of colon
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
  • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
  • G01N2333/4701—Details
  • G01N2333/4713—Plasma globulins, lactoglobulin
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/56—Staging of a disease; Further complications associated with the 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
  • G01N2800/60—Complex ways of combining multiple protein biomarkers for diagnosis

Definitions

• TITLE Progression markers for colorectal adenomas
• the invention relates to the field of oncology. More specifically, the invention relates to methods for typing a colorectal lesion. The invention provides methods and means for differentiating colorectal adenomas and cancer cells from normal cells, based on biomarkers in stool.
• Colorectal cancer is still a major health care problem representing 6.1% percent of all diagnosed cancers worldwide (Bray et al., 2018. CA: Cancer J Clin 68: 394-424). Early detection by way of screening is the most efficient method to reduce the burden of CRC and is now applied in most European countries, Canada, regions of Australia and North and South America and parts of Asia (Navarro et al., 2017. World J Gastroenterol 23: 3632-3642). Such screening aims at detection of disease at a premalignant stage (i.e. colorectal adenoma) or CRC at a curable stage and has been proven to reduce CRC mortality rates (Kerr et al., 2007.
• the FIT test has a high sensitivity for CRC, (i.e. 79%) but the sensitivity for colorectal adenomas (i.e. 31%) is clearly suboptimal leaving room for improvement (Lee et al., 2014. Ann Intern Med 160: 171; de Wijkerslooth et al., 2012. Am J Gastroenterol 107:1570-8; Song and Li, 2016. World J Gastrointest Oncol 8: 793- 800). In this respect, it has been suggested that an increased sensitivity for colorectal adenomas is the best approach to making CRC screening more cost- effective and efficient (Haug et al., 2015. Int J Cancer 136: 2864-74; Imperiale and Kahi, 2018.
• Advanced adenomas defined as adenomas with a size of 310 mm, villous component and/or high-grade dysplasia are currently regarded as an intermediate endpoint for CRC, since these AAs are considered to carry a higher risk of development into a CRC.
• AAs Advanced adenomas
• CAEs cancer associated events
• the aim of this study was to identify specific biomarkers, preferably stool- based biomarkers, that complement or even outperform hemoglobin in the detection of molecularly defined high-risk adenomas and colorectal carcinoma’s (CRCs).
• CRCs molecularly defined high-risk adenomas and colorectal carcinoma’s
• the currently widely used FIT test has a high sensitivity for CRC, but the sensitivity for AA remains suboptimal, therefore additional biomarkers could complement hemoglobin and improve the performance of the current CRC screening.
• CAEs Cancer Associated Events
• adenomas were classified into samples with low risk or high risk for progressing to cancer. Markers panels were identified that significantly outperform HBA1 for the detection of high-risk adenomas and CRCs.
• the invention provides a method for typing an individual suffering from a colorectal lesion, or suspected of suffering therefrom, the method comprising a. providing a sample comprising protein expression molecules from colorectal cells, or suspected to comprise protein expression molecules from colorectal cells; b.
• determining protein expression levels of a set of genes in said sample determining protein expression levels of a set of genes in said sample; and c. typing said sample on the basis of the determined protein expression levels; whereby said set of genes includes at least haptoglobin (Hp).
• Hp haptoglobin
• Said sample preferably is or comprises stool.
• protein expression levels are determined with one or more binding molecules directed against said protein expression molecules of the set of genes.
• a preferred set of genes includes Hp and further comprises at least one of LAMP1, SYNE2, ANXA6, LRG1, RBP4 and FN1, more preferably comprises Hp and at least one of LAMP1, SYNE2 and ANXA6, especially Hp, LAMP1, SYNE2 and ANXA6.
• said typing distinguishes colorectal adenoma cells with a low risk of progression to cancer from colorectal adenoma cells with a high risk of progression to cancer.
• a further preferred set of genes includes Hp and further at least one of LRG1, RBP4 and FN1, especially Hp, LRG1, RBP4 and FN1.
• a preferred set of genes includes HBA1 and/or HBB (together termed hemoglobin of Hb) and further comprises at least one of LAMP1, SYNE2, ANXA6, LRG1, RBP4 and FN1, more preferably comprises Hb and at least one of LAMP1, SYNE 2 and ANXA6, especially Hb, LAMP1, SYNE 2 and ANXA6.
• said typing distinguishes colorectal adenoma cells with a low risk of progression to cancer from colorectal adenoma cells with a high risk of progression to cancer.
• a further preferred set of genes includes Hb and further at least one of LRG1, RBP4 and FN1, especially Hb, LRG1, RBP4 and FN1.
• an enhanced expression level of the protein expression molecules when compared to the expression level of the protein expression molecules in a control sample, is indicative of the presence of high risk adenoma cells, colorectal cancer cells, or both.
• the invention further provides a method comprising (a) providing a sample comprising protein expression molecules from colorectal cells from an individual;
• said set of genes includes at least haptoglobin (Hp) and at least one of LAMP1, SYNE2, ANXA6, LRG1, RBP4 and FN1, preferably Hp, LAMP1, SYNE2 and ANXA6.
• said set of genes includes at least Hb and at least one of LAMP1, SYNE2, ANXA6, LRG1, RBP4 and FN1, preferably Hb, LAMP1, SYNE 2 and ANXA6.
• the invention further provides a method comprising (a) providing a sample comprising extracted protein expression molecules of a set of genes from colorectal cells from an individual; (b) reacting said extracted protein expression molecules with at least one binding molecule that binds to at least one of said protein expression molecules; (c) quantifying a reaction product between said at least one binding molecule and said at least one protein expression molecule; and (e) determining a level of expression of said at least one protein expression molecule, based on the quantified reaction product; whereby said set of genes includes haptoglobin (Hp) and at least one of LAMP1, SYNE2, ANXA6, LRG1, RBP4 and FN1, preferably Hp, LRG1, RBP4 and FN1.
• Hp haptoglobin
• said set of genes includes at least Hb and at least one of LAMP1, SYNE2, ANXA6, LRG1, RBP4 and FN1, preferably Hb, LRG1, RBP4 and FN1.
• the invention further provides a method of assigning treatment to an individual having a high risk colorectal adenoma, comprising (a) typing an individual according to the methods of the invention; (b) identifying an individual that is suspected to have a high risk adenoma; (c) assigning treatment comprising removal of at least part of the high risk adenoma, preferably by polypectomy and/or surgical resection, to the identified individual.
• the invention further provides a kit comprising reagents for directly or indirectly determining a level of expression of protein expression products of haptoglobin (Hp) and at least one of LAMP1, SYNE2, ANXA6, LRG1, RBP4 and FN1, preferably reagents for an
• FIG. 1 Overview of the data analysis approach for the best biomarker panel identification. Feature selection was performed using beta-binomial test (BB- test) in the comparative setting cases vs controls, in particular high-risk adenomas vs healthy controls and high-risk adenomas with CRCs vs healthy controls. Up- regulated proteins were selected using different thresholds for each comparison (see Materials and Methods). Logistic regression with Lasso regularization was applied to built a model based on X features (where X is either two, three or four features). The data analysis approach was evaluated using leave-one-out cross- validation, where feature selection with BB-test and logistic regression with Lasso regularization were repeated. Cross-validated performance of the built models were evaluated with respect to hemoglobin (HBA1) at high specificity levels.
• HBA1 hemoglobin
• FIG. 1 Biomarker panels from logistic regression analysis to identify high- risk adenomas and CRCs.
• ROC curve was obtained from logistic regression predictions from leave-one-out cross-validation analysis. Partial area under the curve (pAUC) was calculated for specificity of 95%-100% and compared to pAUC of hemoglobin to obtain the p-value.
• B Frequency plot of biomarkers occurring in the regression models built during the cross-validation analysis to distinguish between the high-risk adenomas and healthy controls. Four proteins were clearly selected more frequently by the Lasso regularization in the cross-validation analysis.
• FIG. 3 Biomarker panels from logistic regression analysis to identify high- risk adenomas and CRCs.
• B Frequency plot of biomarkers occurring in the regression models built during the cross-validation analysis to discriminate high-risk adenomas and CRCs from healthy controls based on four proteins. Four proteins were clearly selected more frequently by the Lasso regularization in the cross-validation analysis.
• C
• ROC curve was obtained from logistic regression predictions from the leave-one-out cross-validation analysis D. Frequency plot of biomarkers occurring in the regression models built during the cross-validation analysis to discriminate high-risk adenomas and CRCs from healthy controls based on two proteins. The same two proteins were consistently selected in the cross- validation analysis.
• lesion refers to a cancerous growth of epithelial tissue that covers or lines surfaces of the colorectal tract. Said cancerous growth preferably is an adenocarcinoma.
• lesion includes early adenoma, advanced adenoma and colorectal cancer.
• adenoma refers to a benign tumor of epithelial tissue with glandular origin, glandular characteristics, or both. Said adenoma preferably is a colorectal adenoma, also referred to as an adenomatous polyp.
• the term typing refers to assessing presence and/or staging of a lesion.
• the term typing preferably refers to differentiating adenoma’s that are at risk of developing into a carcinoma, including early adenoma and, especially, advanced adenoma.
• the term typing also refers to discriminating colorectal cancer from non-cancerous growth, including normal colorectal tissue. Said typing is intended to provide information to aid in clinical evaluation of patients. The methods of the invention find particular use in choosing appropriate treatment for said patients.
• protein expression molecules refers to protein products of genes, or parts of these products.
• directly conjugated with a detectable label refers to the labeling of the antibody itself with a detectable label.
• the term“indirectly conjugated with a detectable label”, as used herein, refers to the indirect labeling of an antibody, for example using a biotindabelled antibody and a detectable label that is bound to streptavidin, or by using a further antibody that is directed against the indirectly labeled antibody and which further antibody is labeled with a detectable label.
• binding molecule refers to a molecule, preferably a proteinaceous molecule such as a protein that can specifically bind to a target epitope that is present in a protein expression molecule.
• Said binding molecule preferably is an antibody.
• antibody includes classical VH region-containing proteins that may be paired with a light chain variable region (VL).
• VL light chain variable region
• the term antibody also includes synthetic antibody-like molecules or antibody mimics that are known to those skilled in the art such as APTAMERS (Que-Gewirth and Sullenger, 2007. Gene Therapy 14, 283-291); AFFIBODY® molecules (Nord et al., 1995. Prot Eng 8: 601-608), ANTICALINS® (Skerra, 2008. FEBS J. 275: 2677- 2683), and AVIMERS® (Silverman et al., 2005. Nat Biotechnol 23: 1556-1561).
• the term also provides reference to a single domain antibody, a single chain antibody, a nanobody, an unibody, a single chain variable fragment (scFv), a Fd fragment, a Fab fragment and a F(ab')2 fragment.
• a “detectable label” is a label which may be detected and of which the absolute or relative amount and/or location (for example, the location on an array) can be determined.
• the term reference refers to a sample, preferably a stool sample, that comprises protein expression molecules, preferably proteins, from a healthy individual not suffering from a colorectal lesion or from an individual that is known to suffer from a high risk adenoma or from a carcinoma.
• the levels of expression of the protein expression molecules preferably are stored on a computer, or on computer-readable media, to be used in comparisons to the level of expression level data from a sample of an individual.
• specifically binding refers to a binding reaction between an antibody- antigen, or other binding pair, which is determinative of the presence of a protein comprising the antigen in a heterogeneous population of proteins and/or other biologies.
• a specified antibody or functional part thereof binds to a particular antigen and does not bind in a significant amount to other proteins present in the sample.
• polypectomy refers to the partial or complete removal of an adenoma.
• enzyme-linked immunosorbent assay refers to a plate-based assay that is designed for detecting and quantifying antigens such as protein expression molecules.
• a sample from an individual suffering from a colorectal lesion, or suspected to suffer therefrom, comprising protein expression molecules can be obtained in numerous ways, as is known to a skilled person, such as by
• Said sample preferably is or comprises stool from an individual suffering from a colorectal lesion, or suspected to suffer from said lesion.
• a preferred sample is a sample that is obtained from stool by contacting a stool surface, for example with a stick or a brush, and providing a part of the obtained sample in a test tube or on an absorbent surface, for example a test card.
• Said test tube preferably comprises a buffer, for example a stool stabilization buffer such as a buffer comprising phosphate-buffered saline and sodium azide.
• a sample comprising protein expression molecules can be freshly prepared at the moment of isolation of the specimen, or it can be prepared from specimen that have been stored, for example at -20°C, until processing for sample preparation.
• said stool specimen can be stored under conditions that preserve the quality of the protein expression products.
• preservative conditions are fixation, addition of protease inhibitors, addition of reducing agents such as dithiothreitol (DTT) or 2-mercaptoethanol (2-ME), and non-aqueous solutions such as Universal Molecular Fixative (Sakura Finetek USA Inc.; US7138226).
• a stool sample may be mixed with a stool stabilization buffer (for example Exact Sciences, Madison, Wl, USA) after defecation, preferably immediately after defecation, and processed to a final stool:buffer w/v ratio of between 1:1 and 1:2, preferably between 1.2 and 1.7, more preferably about 1:4, within 72 hours, and stored at -80°C until use.
• a stool stabilization buffer for example Exact Sciences, Madison, Wl, USA
• Said sample preferably is pretreated to remove contaminants and/or to increase the concentration of the protein expression molecules. This will result in a lower detection limit and will improve reliability of the methods of the invention.
• a preferred pretreatment method comprises homogenization in a buffer, for example by vortexing, followed by centrifugation, for example for 15 minutes at 16.000 G. After this, the supernatant may be centrifuged for 10 minutes at full speed. Supernatants may be filtered, for example through a 0.22 mM PVDF filter (Merck Millipore, Billerica, MA, USA) and concentrated using a molecular size cut- off filter, for example a 3 kDa cut-off filter (Amicon Ultra, Merck Millipore, Billerica, MA, USA).
• An expression level of a protein expression molecule may be determined by any assay known to a skilled person.
• a level of expression may be determined by polyacrylamide gel electrophoresis, including two dimensional gel electrophoresis, multidimensional protein identification technology, ELISA, bead-based
• immunoassays immuno-PCR using, for example, Thunder-Link® antibody- oligonucleotide conjugation kit (Innova Biosciences. Cambridge UK), surface plasmon resonance, liquid chromatography- tandem mass spectrometry (LC- MS/MS), and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF).
• Thunder-Link® antibody- oligonucleotide conjugation kit Innova Biosciences. Cambridge UK
• LC- MS/MS liquid chromatography- tandem mass spectrometry
• MALDI-TOF matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
• suitable assays are chemo-luminescence assays, fluorescence assays, mass spectrometry, affinity chromatography, Western blotting, Northern blotting, histology and protein expression chips, probes.
• Mass spectrometry is a suitable means of determining a level of expression of a protein.
• a preferred method comprises liquid chromatography coupled to tandem mass spectrometry in positive electrospray ionization mode.
• the LC-MS/MS analysis may be performed, for example by using an I-Class UPLC system connected to a Xevo TQS mass spectrometer Waters (Manchester, UK), or an Q Exactive mass spectrometer (Thermo Fisher).
• a suitable multiplex system for determining an expression level of a protein product is multiple reaction monitoring (MRM), which is a quantitative MS-based approach.
• MRM multiple reaction monitoring
• Said protein expression molecules are preferably detected and quantified using an immunochemical assay, preferably employing binding molecules such as antibodies that specifically bind to a ligand on said protein expression molecules, preferably proteins.
• a protein expression molecule is an antigen for an binding molecule that specifically reacts with said protein expression molecule.
• the binding molecules are preferably coupled to a solid support such as a bead, monolithic material or a multi-well array.
• the binding molecules, preferably antibodies may be coupled directly, or indirectly, for example by coupling of a second binding molecule that specifically recognizes the first binding molecule that binds to a protein expression molecule.
• Indirect coupling may be accomplished, for example, by coupling of antibody-binding molecules such as protein A, protein G, or a mixture of protein A and G to beads, monolithic material or array.
• Direct coupling may be accomplished, for example, by cross-linking, covalently binding or physically adsorbing said binding molecule, preferably antibody, to the solid support.
• a preferred method for determining a level of expression of a protein or multiple proteins includes Enzyme-Linked Immuno Sorbent Assay (ELISA) and Flow Cytometric ImmunoAssay (FCIA).
• ELISA Enzyme-Linked Immuno Sorbent Assay
• FCIA Flow Cytometric ImmunoAssay
• a competition ELISA known amounts of an antigen are immobilized to a surface.
• a sample comprising unknown amounts of said antigen is added, and the antigen is subsequently complexed with a binding molecule that is preferably conjugated, directly or indirectly, to a detectable label such as a colorimetric label, a fluorescent label, a radioactive label or a chemiluminescent label, or an enzyme.
• a detectable label such as a colorimetric label, a fluorescent label, a radioactive label or a chemiluminescent label, or an enzyme.
• detection of the binding molecule that is complexed to the immobilized antigen is accomplished by assessing the conjugated label or enzyme activity via incubation with a substrate to produce a measureable product.
• the amount of label or enzyme activity is inversely proportional to the amount of antigen in the sample.
• a preferred assay is a sandwich ELISA, in which a receptacle is coated with a first binding molecule that is specific to a protein expression molecule, termed “capture binding molecule”, and detection of bound protein expression molecule is accomplished with a second binding molecule, termed“detection binding molecule”. It is preferred that the capture and detection binding molecules do not interfere with each other and can bind simultaneously to said protein expression molecule.
• Said coating of a receptacle or bead may be performed directly or indirectly. Indirect coating may be
• a biotindabeled capture binding molecule that is attached to a linker molecule, for example a U-PLEX Linker (Meso-Scale
• Said receptacle preferably is a multi-well plate, such as a 24 well plate, a 96 well plate, a 192 well plate, or a 384 well plate, in which each of the wells comprises arrayed spots, whereby each of the spots will bind to a specific protein expression molecule.
• Said second binding molecule is preferably directly or indirectly conjugated to a detectable label such as a colorimetric label, a fluorescent label a radioactive label, or a chemiluminescent label, or an enzyme. Detection of the amount of enzyme-conjugated binding molecule is preferably performed by incubation with a substrate to produce a measureable product. As an alternative, turbidimetric assays are preferred, especially for competition ELISAs.
• a detectable label may be a fluorescent, luminescent, chemiluminescent and/or electrochemiluminescent moiety which, when exposed to specific conditions, may be detected.
• a fluorescent label may be exposed to radiation (i.e. light) at a specific wavelength and intensity to cause excitation of the fluorescent label, thereby enabling it to emit detectable fluorescence at a specific wavelength that may be detected.
• the detectable label may be an enzyme which is capable of converting a (preferably undetectable) substrate into a detectable product that can be visualized and/or detected.
• Suitable enzymes include horseradish peroxidase, phosphatase, phosphatase/pyrophosphatase and luciferase.
• the detectable label may be a radioactive label, which may be incorporated by methods known in the art.
• Indirect labeling of a binding molecule may be accomplished, for example, through conjugation of a binding molecule with biotin and reacting biotin with labelled or enzymedinked avidin or streptavidin.
• carbon coated wells may be equipped with electrodes that produce chemical energy when subjected to an electrical charge, such as the Multi- array® and Multi-spot® 96-well plates of Meso-Scale Discovery.
• an electrical charge such as the Multi- array® and Multi-spot® 96-well plates of Meso-Scale Discovery.
• the chemical energy is transformed to emitted light which is measured using a high-resolution CCD camera.
• a level of expression of protein expression molecules is preferably compared with a level of expression of said molecules in a reference.
• Said reference preferably comprises a stool sample from an individual that is known to suffer from a high risk adenoma or a colorectal cancerous growth, or known not to suffer therefrom.
• the level of expression of the at least two protein expression molecules, preferably proteins, in the reference it can be determined whether an individual is likely to have a high risk adenoma, or is likely to suffer from a colorectal cancerous growth. For example, when the reference is sample of a person that is known not to suffer from a high risk adenoma or a colorectal cancerous growth, a difference between the determined level of expression of the at least two protein expression molecules, preferably proteins, might indicate that the individual is suffering from a high risk adenoma or a colorectal cancerous growth.
• a coefficient is determined that is a measure of a similarity or dissimilarity of a sample with said reference.
• a number of different coefficients can be used for determining a correlation between the determined expression levels in a sample from an individual and the comparative levels of expression in said reference.
• Preferred methods are parametric methods which assume a normal distribution of the data.
• a preferred set of protein expression molecules comprises at least expression products from haptoglobin (Hp).
• the haptoglobin gene is identified by references 5141 (HGNC database) and ENSG00000257017 (Ensembl data base).
• the protein expression product is identified by UniProtKB reference number P00738.
• Haptoglobin protein is known to interact with hemoglobin. As a result of hemolysis, hemoglobin accumulates in the kidney and is secreted in the urine. Haptoglobin captures and combines with free plasma hemoglobin to allow hepatic recycling of heme iron and to prevent kidney damage. Haptoglobin also acts as an antioxidant, has antibacterial activity, and plays a role in modulating many aspects of the acute phase response.
• Hp protein was consistently significantly higher abundant in high-risk adenomas and CRCs compared to controls. Hp protein was selected in the three panels whereas hemoglobin (HBA1, HBB or HBD) was not. The fact that levels of hemoglobin were not significantly different in high-risk adenomas compared to controls is in line with the limited sensitivity of FIT for adenomas. Although one would expect that Hp is a marker of blood in stool and, therefore, should not have complementary value to hemoglobin, our data show that Hp may be of added value, especially for the detection of high-risk adenomas.
• FAMP1 Fysosomal Associated Membrane Protein 1
• SYNE2 Spectrin Repeat Containing Nuclear Envelope Protein 2
• ANXA6 Annexin A6
• a preferred set of genes for typing an individual suffering from a colorectal lesion, or suspected of suffering therefrom comprises Hp and at least one of LAMP1, SYNE2, ANXA6, LRG1, RBP4, and FN1.
• Said set of genes preferably comprises Hp and LAMP1, Hp and SYNE2, Hp and ANXA6, Hp, LAMP1 and SYNE2, Hp, SYNE 2 and ANXA6, Hp, LAMP1 and ANXA6, and Hp, SYNE2, LAMP1 and ANXA6.
• Said set of genes preferably is used to distinguish colorectal adenoma cells with a low risk of progression to cancer from colorectal adenoma cells with a high risk of progression to cancer.
• An additional set of genes comprises hemoglobin (Hb, identified by HBA1 and HBB) and at least one of LAMP1, SYNE2, ANXA6, LRG1, RBP4, and FN1, preferably Hb and LAMP1, Hb and SYNE2, Hb and ANXA6, Hb, LAMP1 and SYNE 2, Hb, SYNE 2 and ANXA6, Hb, LAMP1 and ANXA6, and Hb, SYNE 2, LAMP1 and ANXA6.
• Said set of genes preferably is used to distinguish colorectal adenoma cells with a low risk of progression to cancer from colorectal adenoma cells with a high risk of progression to cancer
• HBA1 hemoglobin alpha 1
• HGNC database HGNC database
• ENSG00000206172 Endembl data base
• the protein expression product is identified by UniProtKB reference number P69905.
• HBB hemoglobin beta
• LAMP1 also termed CD 107 Antigen-Like Family Member A
• CD 107 Antigen-Like Family Member A is a lysosome associated membrane protein, which has been implicated in several tumor- promoting activities such as promotion of metastasis, drug resistance and cancer cell survival (Alessandrini, Pezze, and Ciribilli, 2017. Semin Oncol 44:239-253).
• LAMP1 located on chromosome 13q, one of the regions that is commonly gained and that is part of the seven CAEs.
• the LAMP1 gene is identified by references 6499 (HGNC database) and ENSG00000185896 (Ensembl data base).
• the protein expression product is identified by UniProtKB reference number P11279.
• SYNE2 (or Nesprin 2) is a nuclear envelope protein that is involved in regulation of nuclear trafficking. The role of SYNE2 in cancer is yet to be established although there are indications that its presence is pivotal for a DNA damage response (Kelkar et al., 2015).
• SYNE2 Since high-risk adenomas are characterized by chromosomal gains and losses, SYNE2 might be upregulated in response to this DNA damage.
• the SYNE2 gene is identified by references 17084 (HGNC database) and ENSG00000054654 (Ensembl data base).
• the protein expression product is identified by UniProtKB reference number Q8WXH0.
• ANXA6 is present at the cell membrane and in the endosomal compartments, where ANXA6 functions as a multifunctional scaffolding protein. In that position ANXA6 can contribute to many different processes including cancer cell migration and invasion (Grewal et al., 2017). ANXA6 belongs to a family of calcium- dependent membrane and phospholipid binding proteins. The ANXA6 gene is identified by references 544 (HGNC database) and ENSG00000197043 (Ensembl data base). The protein expression product is identified by UniProtKB reference number P08133.
• said set of genes preferably comprises Hp and RBP4, Hp and FN1, Hp and LRG1, Hp, RBP4 and FN1, Hp, FN1 and LRG1, Hp, RBP4 and LRG1, and Hp, FN1, RBP4 and LRG1.
• Said set of genes preferably is used to distinguish high risk adenoma cells, colorectal cancer cells, or both, from normal colorectal cells or from low risk adenoma cells.
• RBP4 has been linked to insulin resistance and has been shown to be present in serum, and was previously described as a potential marker especially for AAs in stool (Bosch et al., 2017. Ann Intern Med 167: 855-866).
• the RBP4 gene is identified by references 9922 (HGNC database) and ENSG00000138207 (Ensembl data base).
• the protein expression product is identified by UniProtKB reference number P02753.
• FN1 is an extracellular matrix protein that is involved in cell adhesion and migration processes, the protein has been shown to be present in serum and has been suggested as a biomarker for hepatocellular carcinoma (Kim et al., 2017).
• the FN1 gene is identified by references 3778 (HGNC database) and ENSG00000115414 (Ensembl data base).
• the protein expression product is identified by UniProtKB reference number P02751.
• LRG1 was reported to be highly upregulated in CRC, both at the mRNA as at the protein level (Zhou et al., 2017, Choi et al., 2013).
• a clear role in tumor development has been established for LRG1, as it stimulates proliferation and inhibition of apoptosis through regulating RUNX1 expression.
• the protein is secreted and may therefore end up in blood or stool passing the tumor.
• increased protein levels of LRG1 in plasma have been reported for colorectal cancer and colon adenoma patients (Ladd et al., 2012, Zhang et al., 2018, Zhou et al., 2017).
• the LRG1 gene is identified by references 29480 (HGNC database) and ENSG00000171236 (Ensembl data base).
• the protein expression product is identified by UniProtKB reference number P02750.
• a further preferred set of genes comprises Hb and RBP4, Hb and FN1, Hb and LRG1, Hb, RBP4 and FN1, Hb, FN1 and LRG1, Hb, RBP4 and LRG1, and Hb, FN1, RBP4 and LRG1.
• Said set of genes preferably is used to distinguish high risk adenoma cells, colorectal cancer cells, or both, from normal colorectal cells or from low risk adenoma cells.
• biomarker proteins have been identified with potential important roles in colorectal carcinogenesis with higher sensitivities for high-risk adenomas and CRCs than HBA1. Our data suggests that these biomarker panels can be used to improve current FIT-based screening approaches.
• the invention further provides a method of assigning treatment to an individual having a high risk colorectal adenoma or a colorectal cancer, comprising (a) typing an individual according to the methods of the invention, (b) identifying an individual that is suspected to have a high risk adenoma; and (c) assigning treatment comprising removal of at least part of the high risk adenoma, preferably by polypectomy and/or surgical resection, to the identified individual.
• biomarkers indicated herein above allows not only the detection of a high risk adenoma, but also enables methods of treating an individual with said high risk adenoma before it progresses into adenocarcinoma. Early diagnosis of high risk adenoma often allows for curative surgical removal of the adenoma, whereas such curative surgical removal may not be possible if diagnosis is delayed.
• Said treatment preferably comprises colonoscopy or polypectomy if said individual is classified as having a high risk adenoma, comprising removal of at least part of the high risk adenoma, preferably complete removal of the high risk adenoma.
• Said removal is preferably followed by an intensified surveillance program according to which a patient from which a high risk adenoma is removed is repeatedly screened at intervals of less than 5 years, preferably less than 3 years, more preferably once yearly.
• a method of assigning or prescribing treatment to an individual of which the adenoma is typed as a high risk adenoma according to the methods of typing of this invention comprising (a) classifying said individual as having a high risk adenoma or as not having a high risk adenoma according to a method of the invention, (b) assigning treatment comprising polypectomy if said individual is classified as having said high risk adenoma.
• a method of assigning or prescribing treatment may further comprise complete removal of the adenoma by surgery Detection of a high risk adenoma using a method of the invention preferably is followed by polypectomy and complete removal of the lesion.
• a method of assigning or prescribing treatment to an individual who is typed as having a colorectal carcinoma according to the methods of typing of this invention comprising (a) classifying said individual as having a colorectal carcinoma or as not having a colorectal carcinoma according to a method of the invention, (b) assigning treatment comprising polypectomy if said individual is classified as having said high risk adenoma and/or (chemo)therapy, such as by assigning or administering 5- fluoruracil (5-FU), preferably in combination with leucovorin, or by assigning or administering capecitabine and/or oxaliplatin and/or irinotecan.
• 5- fluoruracil 5-FU
• Therapeutic agents used to treat a colorectal carcinome include monoclonal antibodies, small molecule inhibitors and chemotherapeutic agents.
• Typical therapeutic monoclonal antibodies include but are not limited to bevacizumab, cetuximab or panitumumab.
• Typical small molecule inhibitors include but are not limited to eriotinib, sorafenib or alisertib. Typical
• chemotherapeutic agents include but are not limited to 5-fluoruracil (5-FU), preferably further in combination with leucovorin, capecitabine, irinotecan and/or oxaliplatin.
• a preferred treatment comprises 5-FU, in combination with leucovorin and oxaliplatinin or 5-FU, in combination with leucovorin and irinotecan.
• a further preferred treatment comprises capecitabine.
• Capecitabine may be used as adjuvant treatment, as monotherapy, or in combination with other agents for advanced or metastatic disease.
• Capecitabine may be used with either irinotecan or oxaliplatin, or used to replace 5-FU in any one of the above indicated combination treatments.
• any of the above indicated combinations comprising 5-FU or capecitabine may be combined with one or more of cetuximab, bevacizumab and panitumumab.
• Combination therapies of, for example, a therapeutic monoclonal antibody and a small molecule inhibitor may be used.
• a therapeutic monoclonal antibody and a small molecule inhibitor may be used.
• any combination of two or more of a monoclonal antibody, a small molecule inhibitor and a chemotherapeutic agent is envisaged.
• the invention further provides a kit for determining whether an individual is having an adenoma with a high risk of progressing to an adenocarcinoma, the kit comprising a device for collecting a sample comprising colorectal cells from said individual, and reagents for directly or indirectly determining a level of expression of at least Hp, preferably Hp and at least one of LAMP1, SYNE2, ANXA6, LRG1, RBP4 and FN1, or Hb and at least one of LAMP1, SYNE 2, ANXA6, LRG1, RBP4 and FN1, in said sample, preferably reagents for an immunochemical assay.
• the kit for performing the method according to the invention may be selected from any suitable assay and data processing apparatus and equipment.
• Said reagents for determining a level of expression of at least Hp preferably are reagents for an immunochemical assay.
• the reagents for determining a level of expression of at least Hp include a receptacle that is coated with antibodies against at least Hp, or monolithic material or microbeads that are coated with antibodies against at least Hp, allowing detection of a level of expression of at least Hp.
• Said receptacle preferably is an array, comprising a solid support and antibodies against at least Hp in an arrayed format that are immobilized on the solid support.
• the solid support is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
• the solid support may be in the form of tubes, beads, discs, silicon chips, microplates, polyvinylidene difluoride (PVDF) membrane, nitrocellulose membrane, nylon membrane, other porous membrane, non-porous membrane (e.g.
• plastic polymer, polymethylmethacrylaat, silicon
• a plurality of polymeric pins or a plurality of microtitre wells, or any other surface suitable for immobilising proteins, antibodies and other suitable molecules and/or conducting an immunoassay.
• well-known techniques such as contact or non-contact printing, masking or photolithography, the location of each spot on said solid support can be defined.
• Said monolithic material or microbeads are preferably coated with antibodies against at least Hp, more preferably against Hp and at least 1 further gene expression product of a gene selected from LAMP1, SYNE2, ANXA6, LRG1, RBP4 and FN1, more preferably against Hp and at least 2 further gene expression products of LAMP1, SYNE2, ANXA6, LRG1, RBP4 and FN1, most preferably against Hp and at least 3 further gene expression products of LAMP1, SYNE2, ANXA6, LRG1, RBP4 and FN1, preferably Hp, LAMP1, SYNE 2 and ANXA6, or Hp LRG1, RBP4 and FN1.
• Said monolithic material or microbeads are preferably coated with antibodies against Hb and at least 1 further gene expression product of a gene selected from LAMP1, SYNE2, ANXA6, LRG1, RBP4 and FN1, more preferably against Hb and at least 2 further gene expression products of LAMP1, SYNE2, ANXA6, LRG1, RBP4 and FN1, most preferably against Hb and at least 3 further gene expression products of LAMP1, SYNE 2, ANXA6, LRG1, RBP4 and FN1, preferably Hb, LAMP1, SYNE2 and ANXA6, or Hb LRG1, RBP4 and FN1.
• Said monolithic material or microbeads coated with antibodies enable simultaneous detection of multiple protein expression molecules.
• the simultaneous analysis is cost effective and amenable to high-throughput/automation.
• the invention further provides a use of a kit according to the invention for use in a method for typing a sample of colorectal adenoma of an individual, preferably for determining presence of absence of an adenoma with a high risk of progressing to an adenocarcinoma.
• FFPE formalin-fixed paraffin embedded
• DNA isolation and copy number identification by low-coverage whole genome sequencing was isolated from FFPE tissues with a column-based method (QIamp DNA microkit, Qiagen, Hilden, Germany) as described before (Buffart et al., 2007. Cell Oncol 29: 351-9), with some adaptations for small lesions. DNA copy number analysis and status for both the adenomas of the study samples as well as the validation samples were reported in a previous publication (Carvalho et al., 2018. Cancer Prev Res (Phila) 11: 403-412). In short, isolated DNA was subjected to low- coverage whole-genome sequencing on the HiSeq 2000 (Illumina) in a 50-bp single- read modus using the Illumina Truseq Nano kit.
• Raw sequence reads were mapped to the human reference genome build GRCh37/hgl9 and data was further analyzed using QDNAseq, CGHcall, CGHregions (Carvalho et al., 2018. Cancer Prev Res (Phila) 11: 403-412).
• Adenomas were characterized for gains of chromosomal arms 8q, 13q, and 20q, and losses of 8p, 15q, 17p, and 18q. If two or more of these CAEs were present the adenoma would be classified as high-risk adenoma (Carvalho et al., 2018. Cancer Prev Res (Phila) 11: 403-412); Hermsen et al., 2002.
• Protein groups were excluded from further analysis if they had missing data for over 15% of the cases, i.e. 13 samples for high- risk adenomas or 80 samples for high-risk adenomas and CRCs.
• Differential protein expression analysis was performed using the beta-binominal test (Pham et al., 2010. Bioinformatics 26: 363-9), log2 fold changes and p-values were obtained. P-values adjusted for multiple hypothesis testing were obtained with the beta-binominal test (Pham et al., 2010. Bioinformatics 26: 363-9), log2 fold changes and p-values were obtained. P-values adjusted for multiple hypothesis testing were obtained with the beta-binominal test (Pham et al., 2010. Bioinformatics 26: 363-9), log2 fold changes and p-values were obtained. P-values adjusted for multiple hypothesis testing were obtained with the beta-binominal test (Pham et al., 2010. Bioinformatics 26: 363-9
• Euclidean distance was used to visualize protein expression profiles. Differential analysis was performed for the following groups and the following thresholds were applied: high-risk adenomas compared to samples without colorectal neoplasia (log2 fold change > 0 and p-value £ 0.1), CRCs and high-risk adenomas compared to samples without colorectal neoplasia (log2 fold change 3 2 and adjusted p-value £ 0.05) to select for proteins higher expressed in cases than in healthy controls.
• Clustering of the proteins higher expressed in cases than controls was performed using hierarchical clustering, where protein abundances were normalized to Z- scores. Subsequently, the Euclidean distance measure was used with ward linkage for samples and complete linkage for proteins.
• the pAUC was compared to pAUC of hemoglobin (HBA1), p-values were obtained with the stratified bootstrap resampling of case/control labels of the individuals with 2000 permutations (Robin et al., 2011. BMC Bioinformatics no. 12:77).
• FIT fluids from the two sample series were analyzed with antibody-based assays.
• Hemoglobin (HBA1 and HBB) and haptoglobin (Hp) protein expression as determined by mass spectrometry were compared to FIT values in the same samples. Missing values were excluded from the analysis. Spearman correlation analysis was performed on normalized spectral counts of HBA1, HBB, Hp and FIT values, correlation coefficients (rho) and p-values were obtained.
• the original four and two protein models were also applied on low-risk adenomas.
• the four protein model classified 6 (11%) out of 56 low-risk adenomas as cases and 50 (89%) as controls, while the two protein model classified 7 (12.5%) low-risk adenomas as cases and 49 (87.5 %) as controls (data not shown).
• Hp The most frequent proteins included in the four protein regression models in the cross-validation procedure were Hp, FRG1, RBP4 and FN1 with frequencies of over 90%, confirming their predictive characteristics and the stability of the model.
• Figure 3C and D When focusing on the overlap of up-regulated proteins in both comparisons and the biomarker panels selected by Lasso regularization, only one protein was present in all panels, Hp. This suggests that Hp might be a crucial component when distinguishing between cases and controls, as it also was one of the most frequent proteins occurring in both analyses.
• Hp was the only protein consistently selected in all three markers panels and therefore we further explored the Hp levels in two FIT cohorts.
• FIT samples were also available for analysis; 16 CRCs, 10 high-risk adenomas, 39 low-risk adenomas and 93 controls.
• Table IB Overview of the proteomics data from the discovery series. Protein abundance per sample as well as results of the differential protein expression analysis were reported. Data sorted for log2 fold change 3 0 and p-value £ 0.05 yielded 61 up-regulated proteins in high-risk adenomas and CRC stool samples.

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