WO2019048588A1 - Panel de biomarqueurs de protéines et d'auto-anticorps mixte pour le diagnostic du cancer colorectal - Google Patents

Panel de biomarqueurs de protéines et d'auto-anticorps mixte pour le diagnostic du cancer colorectal Download PDF

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WO2019048588A1
WO2019048588A1 PCT/EP2018/074087 EP2018074087W WO2019048588A1 WO 2019048588 A1 WO2019048588 A1 WO 2019048588A1 EP 2018074087 W EP2018074087 W EP 2018074087W WO 2019048588 A1 WO2019048588 A1 WO 2019048588A1
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cancer
biomarker
protein
autoantibody
sample
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Hermann Brenner
Hongda Chen
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Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts
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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
    • G01N33/57419—Specifically defined cancers of colon
    • 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
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • 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 present invention pertains to a new method for the diagnosis, prognosis, stratification and/or monitoring of a therapy, of cancer, preferably colorectal cancer, in a patient.
  • the method is based on the determination of the level of a panel of least four biomarker selected from the group consisting of the protein biomarker AREG, GDF-15, FasL, Flt3L, and the autoantibody biomarker TP53 -autoantibody.
  • the new biomarker panel of the invention allows diagnosing and even stratifying various cancer diseases.
  • diagnostic kits for performing the non- invasive methods of the invention are provided.
  • a major step in many aspects of research related to diseases such as cancer is the identification of specific and sensitive biomarkers suitable for the development of effective and improved diagnostic, prognostic and therapeutic modalities.
  • An aim of the present invention is to provide novel biomarkers and biomarker panels for use as novel diagnostic and/or prognostic markers and/or for use in the development of novel therapeutics. Whilst mass spectrometry, shot gun proteomics and DNA/RNA microarray analyses, and deep sequencing have resulted in an increasing list of reported potential tumor biomarkers, very few have found their way into the clinical validation phase and even fewer are used as reliable therapeutic targets or diagnostic markers.
  • CRC colorectal cancer
  • plasma methylated septin 9 is the first and only blood-based test approved by the US Food and Drug Administration (FDA) for CRC screening.
  • FDA US Food and Drug Administration
  • the diagnostic performance of this test is not optimal, with sensitivity for detecting CRC and advanced adenoma of 48.2% and 11.2%, respectively, at 91.5% specificity.
  • Other alleged promising blood biomarkers, such as autoantibodies and microRNAs, were rarely validated in the targeted screening populations.
  • the present invention seeks to provide a novel approach for a simple and minimal invasive but specific and sensitive test system for the diagnosis or monitoring various cancer diseases.
  • the above problem is solved in a first aspect by a method for the diagnosis, prognosis, stratification and/or monitoring of a therapy, of a cancer disease in a subject, comprising the steps of:
  • step (a) Providing a biological sample from the subject, (b) Determining the level (concentration) of at least four biomarker selected from the group consisting of the protein biomarker AREG, GDF-15, FasL, Flt3L, and the autoantibody biomarker TP53 -autoantibody, in the biological sample, wherein a differential level of the at least four biomarkers in the biological sample from the subject as determined in step (b) compared to a healthy control or reference value is indicative for the presence of a cancer disease in the subject.
  • biomarker selected from the group consisting of the protein biomarker AREG, GDF-15, FasL, Flt3L, and the autoantibody biomarker TP53 -autoantibody
  • protein biomarker shall refer in context of the invention to a marker which is a protein molecule, preferably a protein which is not an antibody.
  • autoantibody biomarker shall refer to a specific species of autoantibody used as biomarker. In this case the autoantibody is an indirect sign of an increase of protein expression of a protein biomarker to which the patient or host mounts an immune response reflected in the concentration of level of autoantibodies against said protein.
  • a “diagnosis” or the term “diagnostic” in context of the present invention means identifying the presence or nature of a pathologic condition. Diagnostic methods differ in their sensitivity and specificity.
  • the "sensitivity” of a diagnostic assay is the percentage of diseased individuals who test positive (percent of "true positives”). Diseased individuals not detected by the assay are “false negatives.” Subjects who are not diseased and who test negative in the assay, are termed “true negatives.”
  • the “specificity” of a diagnostic assay is 1 minus the false positive rate, where the "false positive” rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis.
  • prognosis refers to a forecast as to the probable outcome of the disease as well as the prospect of recovery from the disease as indicated by the nature and symptoms of the case. Accordingly, a negative or poor prognosis is defined by a lower post-treatment survival term or survival rate. Conversely, a positive or good prognosis is defined by an elevated post- treatment survival term or survival rate. Usually prognosis is provided as the time of progression free survival or overall survival.
  • stratification refers to the advantage that the method according to the invention renders possible decisions for the treatment and therapy of the patient, whether it is the hospitalization of the patient, the use, effect and/or dosage of one or more drugs, a therapeutic measure or the monitoring of a course of the disease and the course of therapy or etiology or classification of a disease, e.g., into a new or existing subtype or the differentiation of diseases and the patients thereof.
  • stratification means in this context a classification of a colorectal cancer as early or late stage colorectal cancer.
  • monitoring a therapy means for the purpose of the present invention to observe disease progression in a subject who receives a cancer therapy.
  • the subject during the therapy is regularly monitored for the effect of the applied therapy, which allows the medical practitioner to estimate at an early stage during the therapy whether the prescribed treatment is effective or not, and therefore to adjust the treatment regime accordingly.
  • the term “subject” or “patient” refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment. Typically, the terms “subject” and “patient” are used interchangeably herein in reference to a human subject.
  • the term “subject suspected of having cancer” refers to a subject that presents one or more symptoms indicative of a cancer (e.g., a noticeable lump or mass). A subject suspected of having cancer may also have one or more risk factors. A subject suspected of having cancer has generally not been tested for cancer.
  • a "subject suspected of having cancer” encompasses an individual who has received an initial diagnosis (e.g., a CT scan showing a mass) but for whom the subtype or stage of cancer is not known.
  • the term further includes people who once had cancer (e.g., an individual in remission), and people who have cancer and are suspected to have a metastatic spread of the primary tumor.
  • the present invention is also applicable as follow-up care for monitoring a subject for a reoccurrence of the cancer.
  • cancer and “cancer cells” refers to any cells that exhibit uncontrolled growth in a tissue or organ of a multicellular organism.
  • Particular preferred cancers in context of the present invention are selected from colorectal cancer, pancreatic cancer, gastric cancer, breast cancer, lung cancer, prostate cancer, hepatocellular cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, carcinoma, melanoma, leukemia or brain cancer.
  • colonal cancer includes the well-accepted medical definition that defines colorectal cancer as a medical condition characterized by cancer of cells of the intestinal tract below the small intestine (i.e., the large intestine (colon), including the cecum, ascending colon, transverse colon, descending colon, sigmoid colon, and rectum). Additionally, as used herein, the term “colorectal cancer” also further includes medical conditions, which are characterized by cancer of cells of the duodenum and small intestine (jejunum and ileum).
  • gastric cancer or "stomach cancer” refer to cancers of the stomach.
  • carcinomas such as but not limited to, adenocarcinomas, affecting the epithelial cells of the stomach.
  • Stomach cancers may additionally include, for example, sarcomas affecting the connective tissue of the stomach and blastomas affecting the blast tissue of the stomach.
  • pancreatic cancer encompasses benign or malignant forms of pancreatic cancer, as well as any particular type of cancer arising from cells of the pancreas (e.g., duct cell carcinoma, acinar cell carcinoma, papillary carcinoma, adenosquamous carcinoma, undifferentiated carcinoma, mucinous carcinoma, giant cell carcinoma, mixed type pancreatic cancer, small cell carcinoma, cystadenocarcinoma, unclassified pancreatic cancers, pancreatoblastoma, and papillary-cystic neoplasm, and the like.).
  • duct cell carcinoma acinar cell carcinoma
  • papillary carcinoma adenosquamous carcinoma
  • undifferentiated carcinoma mucinous carcinoma
  • giant cell carcinoma giant cell carcinoma
  • mixed type pancreatic cancer small cell carcinoma, cystadenocarcinoma, unclassified pancreatic cancers, pancreatoblastoma, and papillary-cystic neoplasm, and the like.
  • biological sample refers to a sample that was obtained and may be assayed for any one of the biomarkers as disclosed with the present invention, or their gene expression.
  • the biological sample can include a biological fluid (e.g., blood, cerebrospinal fluid, urine, plasma, serum), tissue biopsy, and the like.
  • the sample is a tissue sample, for example, tumor tissue, and may be fresh, frozen, or archival paraffin embedded tissue.
  • Preferred samples for the purposes of the present invention are bodily fluids, in particular blood or plasma samples.
  • a “biomarker” or “marker” in the context of the present invention refers to an organic biomolecule, particularly a polypeptide, which is differentially present in a sample taken from subjects having a certain condition as compared to a comparable sample taken from subjects who do not have said condition (e.g., negative diagnosis, normal or healthy subject, or non- cancer patients, depending on whether the patient is tested for cancer, or metastatic cancer).
  • a marker can be a polypeptide or polysaccharide (having a particular apparent molecular weight) which is present at an elevated or decreased level in samples of cancer patients compared to samples of patients with a negative diagnosis.
  • determining the level of a biomarker in a sample, control or reference, as described herein shall refer to the quantification of the presence of said biomarkers in the testes sample.
  • concentration of the biomarkers in said samples may be directly quantified via measuring the amount of protein/polypeptide/polysaccharide as present in the tested sample.
  • quantify the amount of biomarker indirectly via assessing the gene expression of the encoding gene of the biomarker, for example by quantification of the expressed mR A encoding for the respective biomarker.
  • Level in the context of the present invention is therefore a parameter describing the absolute amount of a biomarker in a given sample, for example as absolute weight, volume, or molar amounts; or alternatively "level” pertains to the relative amounts, for example and preferably the concentration of said biomarker in the tested sample, for example mo 1/1, g/1, g/mol etc. In preferred embodiments the "level” refers to the concentration of the tested biomarkers in g/1.
  • certain biomarkers as disclosed herein may also be significantly decreased in the event of a cancer disease in a subject.
  • the method of the herein disclosed invention is performed noninvasive, such as an in vitro or ex vivo method.
  • diagnostic methods are non-invasive the term "providing a biological" sample shall preferably not be interpreted to include a surgical procedure conducted at the subject.
  • Preferred embodiments of the present invention pertain to panels of a plurality of biomarkers as identified herein for the diagnostic purposes as described.
  • the advantage of combing the biomarkers, in particular the combination of protein biomarkers and autoantibody biomarkers, as disclosed herein, is an increased sensitivity and/or specificity of the diagnostic assays.
  • a preferred embodiment of the invention pertains to the herein disclosed method wherein step (b) comprises determining the level of at least four or preferably all five, biomarkers in the biological sample. Most preferred is that at least five biomarkers are used.
  • the level of at least CEA, AREG, and GDF-15, in the biological sample is determined.
  • one or more additional biomarkers known to be useful in the diagnosis of the disease may be tested.
  • the analysis of the marker panel in step (b) of the diagnostic method of the invention is characterized in that the tested marker panel has an apparent area under the curve (AUC) at 95% confidence interval (CI) of at least 60%, preferably at least 65% or more preferably at least 70%.
  • AUC apparent area under the curve
  • CI 95% confidence interval
  • the panel of the invention may be characterized by a sensitivity of at least 75%, preferably at least 80%>, and a specificity of at least 40%>, preferably at least 50%, more preferably at least 60%.
  • the CI and specificity/sensitivity of the marker panels are as disclosed in the example section.
  • the biomarkers of the invention are preferably protein biomarkers and/or autoantibody biomarkers.
  • the biomarker panel as disclosed herein is particular useful in a cancer screening setting.
  • Cancer screening in the herein disclosed invention shall refer to a procedure where a subject is for which not diagnosis was established is tested for the presence of the cancer disease. This shall not be interpreted to exclude the use of the biomarker of the invention for a diagnostic of a subject that was already diagnosed to suffer from a cancer disease.
  • Non limiting examples for such an application are confirmation of a diagnosis, monitoring or treatment success or monitoring reoccurrence of a cancer in a subject that already received a treatment and wherein cancer is in remission or was cured.
  • a threshold value may be obtained by performing the assay method on samples obtained from a population of patients having a certain type of cancer, and from a second population of subjects that do not have cancer.
  • a population of patients all of which have, for example, ovarian cancer, may be followed for the time period of interest (e.g., six months following diagnosis or treatment, respectively), and then dividing the population into two groups: a first group of subjects that progress to an endpoint (e.g., recurrence of disease, death); and a second group of subjects that did not progress to the end point.
  • endpoints include, but are not limited to, 5-year mortality rates or progression to metastatic disease.
  • ROC Receiver Operating Characteristic curves
  • a threshold is selected, above which (or below which, depending on how a marker changes with the disease) the test is considered to be “positive” and below which the test is considered to be “negative.”
  • the area under the ROC curve (AUC) is a measure of the probability that the perceived measurement may allow correct identification of a condition.
  • thresholds may be established by obtaining an earlier marker result from the same patient, to which later results may be compared.
  • the individuals act as their own "control group.”
  • markers that increase with disease severity or prognostic risk an increase over time in the same patient can indicate a worsening of disease or a failure of a treatment regimen, while a decrease over time can indicate remission of disease or success of a treatment regimen.
  • thresholds or reference values may be determined. This can be the case in so-called “tertile,” “quartile,” or “quintile” analyses.
  • the "disease” and “normal” groups (or “low risk” and “high risk”) groups can be considered together as a single population, and are divided into 3, 4, or 5 (or more) "bins” having equal numbers of individuals. The boundary between two of these "bins” may be considered “thresholds.”
  • a risk (of a particular diagnosis or prognosis for example) can be assigned based on which "bin” a test subject falls into.
  • said sample is selected from the group consisting of body fluids or tissue, preferably wherein said body fluid sample is a blood sample, more preferably a plasma or serum sample.
  • the level of said at least one biomarker in said sample is determined by means of a nucleic acid detection method or a protein detection method.
  • nucleic acid detection methods are only applicable where an expressed protein is the biomarker.
  • all means shall be comprised by the present invention which allow for a quantification of the expression of any one of the herein disclosed biomarker. Therefore also promoter analysis and procedures assessing the epigenetic status of a gene locus encoding a protein biomarker of the invention are comprised by the herein described invention.
  • the level of said at least one biomarker in said sample is determined by means of a detection method selected from the group consisting of mass spectrometry, mass spectrometry immunoassay (MSIA), antibody-based protein chips, 2-dimensional gel electrophoresis, stable isotope standard capture with anti-peptide antibodies (SISCAPA), high-performance liquid chromatography (HPLC), western blot, cytometry bead array (CBA), protein immuno- precipitation, radio immunoassay, ligand binding assay, and enzyme-linked immunosorbent assay (ELISA), preferably wherein said protein detection method is ELISA.
  • a detection method selected from the group consisting of mass spectrometry, mass spectrometry immunoassay (MSIA), antibody-based protein chips, 2-dimensional gel electrophoresis, stable isotope standard capture with anti-peptide antibodies (SISCAPA), high-performance liquid chromatography (HPLC), western blot, cytometry bead array (CBA), protein immuno- precipitation, radio immunoa
  • an immunological capture assay using a protein or protein fragment is preferred.
  • the autoantibody is detected by detecting the binding of the autoantibody to its respective antigen, or to a fragment of the antigen which contains the binding epitope.
  • the autoantibody biomarker is TP53 -autoantibody
  • said capturing protein is TP53 protein, or an antigenic fragment thereof, preferably wherein the antigenic fragment comprises an epitope to which said TP53 -autoantibody is capable to bind.
  • kits for aiding a diagnosis of cancer wherein the kits can be used to detect the biomarkers of the present invention.
  • the kits can be used to detect any one or combination of biomarkers described above, which biomarkers are differentially present in samples of a patient having the cancer and healthy patients.
  • the kits of the invention have many applications.
  • the kits can be used to differentiate if a subject has the cancer, or has a negative diagnosis, thus aiding a cancer diagnosis.
  • the kits can be used to identify compounds that modulate expression of the biomarkers in in vitro cancer cells or in vivo animal models for cancer.
  • the kit can further comprise instructions for suitable operational parameters in the form of a label or a separate insert.
  • the kit may have standard instructions informing a consumer how to wash the probe after a sample of plasma is contacted on the probe.
  • kits comprises (a) an antibody that specifically binds to a marker; and (b) a detection reagent.
  • a kit can be prepared from the materials, and the previous discussion regarding the materials (e.g., antibodies, detection reagents, immobilized supports, etc.) is fully applicable to this section and need not be repeated.
  • the kit may optionally further comprise a standard or control information so that the test sample can be compared with the control information standard to determine if the test amount of a marker detected in a sample is a diagnostic amount consistent with a diagnosis of cancer.
  • the kit of the invention is a diagnostic kit for performing a method in accordance with the present invention comprising means for quantifying the level of said at least one biomarker.
  • the kit of the invention comprises means for quantifying a protein biomarker selected from AREG, GDF-15, FasL, and Flt3L.
  • Such means for quantifying is for example at least one antibody, preferably wherein the antibody is a monoclonal antibody, such as a monoclonal antibody that specifically binds to any of the aforementioned biomarkers.
  • Such antibodies are known in the art and commercially available.
  • the diagnostic kit of the invention may contain means for detection of the presence or absence, and quantification thereof, of the autoantibody biomarker TP53 autoantibody.
  • Figure 1 STAandards for the Reporting of Diagnostic accuracy studies (STARD) diagram showing the selection of study participants enrolled in the BLITZ study in 2005-2015 and the analysis scheme.
  • STARD Diagnostic accuracy studies
  • Figure 2 Comparison of receiver operating characteristics curves of the four-marker panel and five-marker panel for detecting: a) colorectal cancer vs. controls free of neoplasm; b) advanced adenomas vs. controls free of neoplasm.
  • the BLITZ study (Beg toode Evaluierung innovativer Testtechnisch Kunststoff Darmkrebsfruerkennung) is an ongoing cohort of participants attending screening colonoscopy in Germany. Detailed information on the BLITZ study has been reported elsewhere (Chen H, Zucknick M, Werner S, Knebel P, Brenner H. Head-to-Head Comparison and Evaluation of 92 Plasma Protein Biomarkers for Early Detection of Colorectal Cancer in a True Screening Setting. Clin Cancer Res 2015;21 :3318-26; Brenner H, Tao S, Haug U. Low-dose aspirin use and performance of immunochemical fecal occult blood tests. JAMA 2010;304:2513-20.).
  • CRC stage was classified according to the UICC (Union for International Cancer Control) TNM (tumor-node-metastasis) stage classification (7th version). Participants of screening colonoscopy were classified according to the most advanced finding reported in the colonoscopy and/or histology report.
  • Advanced adenomas were defined as adenomas with at least one of the following features: 1) high grade dysplasia (HGD); 2) villous or tubular- villous architecture; 3) size >10 mm. Relevant information was extracted from colonoscopy and hospital records by two research assistants independently who were blinded with respect to the blood test results.
  • Protein profiling Protein profiling (Protein Biomarker)
  • the raw data of the protein profiling were firstly normalized following the standard protocol from the manufacturer and using the Olink Wizard of GenEx software (MultiD, Goteborg, Sweden). For each data point, the raw quantification cycle value (Cq-value, in log2 scale) was exported from the Fluidigm Real-Time PCR Analysis Software. The Cq-value is defined as the calculated cycle number at which the PCR product crosses a threshold of detection and is used to represent the expression levels of respective proteins in the present study. The first step of normalization was to subtract the raw Cq-value for the extension control for the corresponding sample in order to correct for technical variation.
  • dCq-value The calculated Cq-values (dCq-value) were further normalized against the negative control determined in the measurement, which yielded ddCq-values (hereafter: Cq-value, in log2 scale) and could be used for further analyses.
  • Limit of detection (LOD) was defined as the mean value of the three negative controls plus 3 calculated standard deviations. 30 samples with invalid test results were excluded from this analysis. Missing data and data with a value lower than LOD were replaced with LOD in the following statistical analyses.
  • the discovery set samples included CRC cases recruited in the clinical setting and 118 randomly selected controls free of neoplasm from the BLITZ study ( Figure 1).
  • the validation set was defined in such a way that it represents a true screening setting, i.e., only participants from the BLITZ study were included.
  • Cq-value The plasma protein levels (Cq-value) were first compared between CRC cases and neoplasm- free controls in the discovery set samples and validation set samples using Wilcoxon Rank Sum Test (hereafter: Wilcoxon test).
  • Wilcoxon test The Benjamini & Hochberg method was additionally employed to correct for multiple testing.
  • a multi-marker algorithm was derived by applying the Lasso logistic regression model based on significant bio markers identified in the discovery set samples.
  • a second prediction algorithm was built by combining the measurements of the selected protein biomarkers from the Lasso logistic regression model with TP53 autoantibody measurements using logistic regression. Both prediction algorithms were further validated using receiver operating characteristics (ROC) curves in the validation set. Areas under the curve (AUCs) and sensitivities at cutoffs yielding 80% and 90%> specificity, respectively, and their 95% CIs of the multi-marker algorithms were calculated and reported.
  • ROC receiver operating characteristics
  • AUCs Areas under the curve
  • sensitivities at cutoffs yielding 80% and 90%> specificity, respectively, and their 95% CIs of the multi-marker algorithms were calculated and reported.
  • the inventors conducted subgroup analyses on the diagnostic performance of the multi-marker algorithms according to sex and age ( ⁇ 65 years vs. >65 years) and cancer stage in the validation set. Statistical analyses were performed with the
  • Figure 1 provides the STAandards for the Reporting of Diagnostic accuracy studies (STARD) diagram showing the selection of study participants enrolled in the BLITZ study in 2005- 2015 and also the scheme of analysis.
  • the discovery set included 226 clinically recruited CRC cases and 118 controls free of colorectal neoplasms.
  • the validation set included 41 CRC cases, 106 participants with advanced adenomas and 107 controls free of colorectal neoplasms all of whom were recruited in the screening setting.
  • Adenoma > 1cm - - - 36 (52.8) -
  • CRC colorectal cancer
  • HGD high-grade dysplasia
  • UICC TNM Union for International
  • Table 1 shows the distribution of study population characteristics of the discovery set and the validation set.
  • CRC cases were on average a few years older than controls free of neoplasm and advanced adenomas.
  • the proportion of men was somewhat higher in the CRC groups and in the advanced adenoma group than in the control groups.
  • Approximately half of the CRC cases were diagnosed in early (I or II) stages.
  • a slightly higher proportion of CRCs was diagnosed at early stage (stage I/II) for the discovery set than for the validation set (57.9% vs. 41.4%). More cancer patients had their tumor located in the colon than in the rectum.
  • CRC colorectal cancer
  • GDF-15 Growth differentiation factor 15
  • AREG Amphiregulin
  • TRAILR-2 TNF-related apoptosis-inducing ligand receptor-2
  • IL-6 Interleukin-6
  • AM Adrenomedullin
  • HE4 WAP four-disulfide core domain protein 2; TNF-R2, Tumor necrosis factor receptor-2; ILT3,
  • Immunoglobulin-like transcript 3 CEA, Carcinoembryonic antigen; CXCL9, C-X-C motif chemokine 9;
  • TNFR-1 Tumor necrosis factor receptor-1
  • HGF Hepatocyte growth factor
  • the inventors used the lasso logistic regression models to construct a multi-marker prediction algorithm based on the 39 significant bio markers identified in the discovery set.
  • the following 4 proteins were selected in the algorithm: growth differentiation factor 15 (GDF- 15), am hiregulin (AREG), Fas antigen ligand (FasL) and Fms-related tyrosine kinase 3 ligand (Flt3L).
  • the four biomarker panel constitutes Example 1 of the present invention.
  • Another prediction model combining these 4 proteins with TP53 autoantibody was further constructed.
  • the five biomarker panel constitutes Example 2 of the present invention.
  • the apparent AUCs of the 4-marker algorithm and 5-marker algorithm for discriminating CRC vs. controls free of neoplasm were 0.87 (95% CI, 0.83-0.90) and 0.89 (95% CI, 0.85-0.92), respectively.
  • AUC area under the curve
  • CRC colorectal cancer
  • 95% CI 95% confidence interval
  • 4 protein panel including GDF-15, AREG, Fas and Flt3L.
  • Table 3 and Figure 2 show the comparison of the two prediction algorithms in detecting CRC and its precursors in the validation set.
  • the AUC of the 4-protein panel for discriminating CRC versus controls free of neoplasm was 0.81 (95% CI, 0.73-0.88).
  • Adding TP53 autoantibody to the four-protein panel conferred a modest improvement in terms of AUC (0.82, 95%) CI, 0.74-0.90), but suprisingly strong improvement could be observed at the left side of the ROC curve.
  • the sensitivity of the four- and the five-marker algorithm for detecting CRC were 53.6%> (95%> CI, 26.8-70.7%)) and 56.4% (95% CI, 38.4-71.8%), respectively, and at cutoffs yielding 80% specificity, the sensitivity of the four- and the five-marker algorithm for detecting CRC were 63.4% (95% CI, 48.8-82.9%) and 66.7% (95% CI, 48.7-82.1%), respectively.
  • the inventors evaluated the diagnostic performance of 92 plasma proteins and serum TP53 autoantibodies for detecting CRC and its precursors in a head-to-head manner using a large set of samples. Twelve protein biomarkers showed significantly higher expression levels in CRC patients than in controls free of neoplasms in both the discovery set and a validation set that was entirely derived from a true screening setting. Moreover, a five- marker panel including GDF-15, AREG, FasL, Flt3L and TP53 autoantibody (Example 2) was constructed and validated.
  • the AUCs of the five-marker panel for detecting CRC and advanced adenomas were 0.82 (95% CI, 0.74-0.90) and 0.60 (95% CI, 0.74-0.90), respectively.
  • the panel showed similar diagnostic performance for detecting early and late stage CRCs.
  • GDF-15 also known as macrophage inhibitory cytokine- 1
  • AREG a mediator of systemic inflammatory response and has been reported to be related to various types of cancer.
  • Advanced adenoma is the most important precursor of CRC, which a substantial risk of development into CRC in the long run. Early detection and removal of these precancerous lesions could therefore reduce the risk of CRC occurrence. To date, it is still a major challenge to detect advanced adenomas using blood-based tests, and most studies found very poor diagnostic performance for this outcome. Although some candidates, such as miRNA- 135b and a panel of BAG4, IL6ST and CD44, were reported in some studies to present good sensitivity for detecting advanced adenomas, these findings were either derived from studies having limited sample size or using clinically identified cases, thus requiring further independently validation in larger screening populations.
  • the panel of Example 2 exhibited comparable diagnostic performance compared to the plasma methylated septin9, the only US FDA approved blood based test for CRC screening and therefore presents another diagnostic blood based option for patients in the future.
  • the sensitivity of methylated septin 9 for detecting CRC and advanced adenomas were reported to be 48.2% and 11.2%, respectively, at a specificity of 91.5%>.
  • the sensitivities of the five-marker panel for detecting CRC and advanced adenomas were 56.4% and 20.7%, respectively (not reported in the results section), demonstrating better diagnostic performance.
  • advantageous aspects of the invention include the adopted a two-step approach, with biomarker discovery and subsequent validation in an independent sample set.
  • the validation set consisted of prediagnostic samples from a large cohort of participants attending screening colonoscopy, therefore representing the target population for CRC screening.
  • both CRC and its precursors were included in the validation set, therefore rendering a thorough overview of the diagnostic potential of all examined biomarkers and the multi-marker panels.
  • a large number of markers were tested simultaneously using state-of-the-art techniques, making a direct comparison of the diagnostic performance of all tested markers possible.

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Abstract

La présente invention concerne une nouvelle méthode pour le diagnostic, le pronostic, la stratification et/ou la surveillance d'une thérapie du cancer, de préférence du cancer colorectal, chez un patient. La méthode repose sur la détermination du niveau d'un panel d'au moins quatre biomarqueurs choisis dans le groupe constitué par le biomarqueur de protéine AREG, GDF-15, FasL, Flt3L, et le biomarqueur d'auto-anticorps de l'auto-anticorps TP53. Le nouveau panel de biomarqueurs selon l'invention permet de diagnostiquer et même de stratifier plusieurs maladies cancéreuses. L'invention concerne également des kits de diagnostic permettant la mise en œuvre des méthodes non invasives de l'invention.
PCT/EP2018/074087 2017-09-07 2018-09-07 Panel de biomarqueurs de protéines et d'auto-anticorps mixte pour le diagnostic du cancer colorectal WO2019048588A1 (fr)

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WO2020225426A1 (fr) * 2019-05-08 2020-11-12 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Examen de dépistage du cancer colorectal et procédé de détection précoce
WO2021185982A1 (fr) * 2020-03-19 2021-09-23 Advanced Marker Discovery S.l. Signature protéque pour cribler une population globale pour un cancer colorectal et/ou un stade précancéreux correspondant
WO2021206523A1 (fr) * 2020-04-10 2021-10-14 (주)바이오니아 Composition utilisant un échantillon d'urine pour le diagnostic d'une maladie rénale
CN114924075A (zh) * 2022-05-26 2022-08-19 郑州大学第一附属医院 基于聚焦阵列蛋白芯片筛选用于贲门腺癌诊断的生物标志物及其应用
CN116519954A (zh) * 2023-06-28 2023-08-01 杭州广科安德生物科技有限公司 一种结直肠癌检测模型构建方法、系统及生物标志物

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020225426A1 (fr) * 2019-05-08 2020-11-12 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Examen de dépistage du cancer colorectal et procédé de détection précoce
WO2021185982A1 (fr) * 2020-03-19 2021-09-23 Advanced Marker Discovery S.l. Signature protéque pour cribler une population globale pour un cancer colorectal et/ou un stade précancéreux correspondant
WO2021206523A1 (fr) * 2020-04-10 2021-10-14 (주)바이오니아 Composition utilisant un échantillon d'urine pour le diagnostic d'une maladie rénale
CN114924075A (zh) * 2022-05-26 2022-08-19 郑州大学第一附属医院 基于聚焦阵列蛋白芯片筛选用于贲门腺癌诊断的生物标志物及其应用
CN116519954A (zh) * 2023-06-28 2023-08-01 杭州广科安德生物科技有限公司 一种结直肠癌检测模型构建方法、系统及生物标志物
CN116519954B (zh) * 2023-06-28 2023-10-27 杭州广科安德生物科技有限公司 一种结直肠癌检测模型构建方法、系统及生物标志物

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