WO2019076949A1 - Moyen et procédés de classification des cancers colorectaux - Google Patents

Moyen et procédés de classification des cancers colorectaux Download PDF

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Publication number
WO2019076949A1
WO2019076949A1 PCT/EP2018/078340 EP2018078340W WO2019076949A1 WO 2019076949 A1 WO2019076949 A1 WO 2019076949A1 EP 2018078340 W EP2018078340 W EP 2018078340W WO 2019076949 A1 WO2019076949 A1 WO 2019076949A1
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cpg sites
methylation status
subject
cpg
methylation
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PCT/EP2018/078340
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English (en)
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Barbara BURWINKEL
Melanie MAIERTHALER
Dominic EDELMANN
Hermann Brenner
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Deutsches Krebsforschungszentrum
Universität Heidelberg
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Publication of WO2019076949A1 publication Critical patent/WO2019076949A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers

Definitions

  • the present invention relates to a method for determining a survival probability of a subject suffering from colorectal cancer comprising a) detecting the methylation status of at least two CpG sites related to at least two CpG sites selected from cgl6336556, cgl4270346, cg05646575, cgl7431888, cgl2510999, cg00832644, cgl 1056055, cg08804626, cgl4983135, cg22522598, cgl9184885, cg08729279, cgl0758824, cgl8195165, cg08617020, cg23750514, cgOl 131395, cgl8736676, cgl9340296, and cgl6399624 in a sample of said subject and, b) based on the methylation status detected in step a), determining the survival probability of said subject; and
  • Colorectal cancer is the third most common cancer worldwide accounting for 1.36 million new cases annually (Ferlay et al, International journal of cancer Journal international du cancer 2015;136(5):E359-86). The five-year survival rate of patients is highly dependent on the stage of the disease (Siegel et al; CA Cancer J Clin 2012;62(4):220-41).
  • DNA methylation predominantly defined as an addition of a methyl-group at cytosine residues located adjacent to guanine bases (CpG dinucleotides), is one of the major epigenetic mechanisms, important in many physiological and pathophysiological processes (Egger et al; Nature 2004;429(6990):457-63). Since many years, the dysregulation of DNA methylation has been known to have a key role in cancer development and progression (Feinberg & Tycko; Nat Rev Cancer 2004;4(2): 143-53).
  • CIMP CpG island methylator phenotype
  • the present invention relates to a method for determining a survival probability of a subject suffering from colorectal cancer comprising
  • step b) based on the methylation status detected in step a), determining the survival probability of said subject.
  • the terms “have”, “comprise” or “include” or any arbitrary grammatical variations thereof are used in a non-exclusive way. Thus, these terms may both refer to a situation in which, besides the feature introduced by these terms, no further features are present in the entity described in this context and to a situation in which one or more further features are present.
  • the expressions “A has B”, “A comprises B” and “A includes B” may both refer to a situation in which, besides B, no other element is present in A (i.e. a situation in which A solely and exclusively consists of B) and to a situation in which, besides B, one or more further elements are present in entity A, such as element C, elements C and D or even further elements. It will be understood that any component defined herein as being included may preferably be explicitly excluded from the claimed invention by way of proviso or negative limitation.
  • the term "about” relates to the indicated value with the commonly accepted technical precision in the relevant field, preferably relates to the indicated value ⁇ 20%, more preferably ⁇ 10%, most preferably ⁇ 5%.
  • the term “essentially” indicates that deviations having influence on the indicated result or use are absent, i.e. potential deviations do not cause the indicated result to deviate by more than ⁇ 20%, more preferably ⁇ 10%>, most preferably ⁇ 5%.
  • “consisting essentially of means including the components specified but excluding other components except for materials present as impurities, unavoidable materials present as a result of processes used to provide the components, and components added for a purpose other than achieving the technical effect of the invention.
  • a composition consisting essentially of a set of components will comprise less than 5% by weight, more preferably less than 3% by weight, even more preferably less than 1%, most preferably less than 0.1% by weight of non-specified component(s).
  • the term "essentially identical" indicates a %identity value of at least 80%, preferably at least 90%, more preferably at least 98%, most preferably at least 99%). As will be understood, the term essentially identical includes 100% identity.
  • nucleic acid sequences and amino acid sequences are noted according to conventional notation, Thus, nucleic acid sequences noted in the direction 5' to 3', and amino acid sequences in the direction N-terminal to C-terminal.
  • the method for determining a survival probability of the present invention preferably, is an in vitro method. Moreover, it may comprise steps in addition to those explicitly mentioned above. For example, further steps may relate, e.g., to obtaining a sample for step a), deriving recommendations for further proceeding and/or providing treatment after obtaining the result of step b), and/or further steps as specified herein below.
  • the method preferably, comprises further including at least one, more preferably at least three, more preferably at least five, most preferably all of the standard clinical factors gender, age, tumor stage, tumor location, smoking behavior and MSI status in the determination, preferably as specified herein in the Examples. Moreover, one or more of said steps may be performed by automated equipment.
  • the term "survival probability” relates to the probability that a subject will be alive during certain period of time.
  • a survival probability is also a measure for a mortality risk, i.e. for the probability that said subject dies within the indicated period of time.
  • said period of time is at most 6 years, more preferably at most 5 years, more preferably at most 50 months, even more preferably at most 40 months, most preferably at most 30 months.
  • the survival probability may be a favorable survival probability, i.e. a survival probability indicating a low probability for dying within one of the aforesaid time frames.
  • the survival probability for the aforesaid time frames in case a favorable survival probability is determined is at least 0.75, more preferably at least 0.8, still more preferably at least 0.9, most preferably at least 0.95.
  • the survival probability may be an unfavorable survival probability, i.e. a survival probability indicating a decreased probability for surviving one of the aforesaid time frames.
  • the survival probability for the aforesaid time frames in case an unfavorable survival probability is determined is at most 0.74, more preferably at most 0.7, even more preferably at most 0.7, most preferably at most 0.65.
  • determining a survival probability of a subject relates to determining the probability according to which the subject will survive the aforesaid time frame, which also is a measure for the probability to die within one of the aforesaid time frames.
  • the aforesaid time frames are calculated from the time, preferably the day, the sample is obtained from the subject.
  • the subject preferably, is a subject known to suffer from cancer, preferably colorectal cancer.
  • the method of the present invention does not provide diagnosis that a subject is, at the time of assessment, afflicted with disease, in particular colorectal cancer.
  • determining a survival probability is not diagnosing a specific disease, more preferably is not diagnosing disease.
  • the method for determining a survival probability is not required to be performed by a medical practitioner, more preferably is not performed by a medical practitioner.
  • the result of the method of the present invention is not a diagnosis of disease.
  • an unfavorable survival probability is determined according to the method of the present invention
  • the subject and/or the counseling medical practitioner may decide to or recommend to perform life-style changes in order to improve its survival probability; also, treatment methods, in particular aggressive treatment methods, may be recommended, e.g. surgery, high-dose chemotherapy and/or high-dose radiotherapy.
  • treatment methods in particular aggressive treatment methods, may be recommended, e.g. surgery, high-dose chemotherapy and/or high-dose radiotherapy.
  • the subject is recommended to be treated with at least one of surgery, chemotherapy, and radiotherapy; also preferably, in case a favorable survival probability is determined, the subject is recommended to be treated by avoiding chemotherapy and/or radiotherapy.
  • detecting an unfavorable survival probability preferably, provides an indication that a subject has an increased probability to, preferably within the time frames as specified above, experience severe aggravation of disease, preferably at least of one of the diseases as specified herein.
  • detecting an unfavorable survival probability preferably, provides an indication that a subject has an increased probability to, preferably within the time frames as specified above, die from disease, preferably at least one of the diseases as specified herein.
  • the term "subject”, as used herein, relates to an animal, preferably a mammal, and, more preferably, a human.
  • the subject according to the present invention is a subject of at least 40 years of age, more preferably at least 50 years of age, even more preferably at least 60 years of age, most preferably at least 65 years of age.
  • the subject has been diagnosed with cancer, more preferably colorectal cancer, even more preferably with non- metastatic colorectal cancer.
  • the term "apparently healthy subject” relates to a subject not known to suffer from colorectal cancer, preferably not suspected to suffer from colorectal cancer based on physical examination, more preferably not showing any symptom of disease, even more preferably not known to suffer from cancer and not showing any symptoms of cancer based on physical examination.
  • the subject and the apparently healthy subject preferably are corresponding subjects from the same species, preferably from the same race.
  • sample refers to a sample of a body fluid, to a sample of separated cells or to a sample from tissue of the subject; preferably, the term refers to a tumor cell- comprising sample of a body fluid, to a sample of separated tumor cells (e.g. circulating/dissimilated tumor cells in peripheral blood) or to a sample from tumor tissue of the subject.
  • Samples of body fluids can be obtained by well known techniques and include, preferably, samples of blood, plasma, serum, or urine.
  • Tissue or organ samples may be obtained from any tissue or organ by, e.g., biopsy.
  • Separated cells may be obtained from the body fluids or the tissues or organs by separating techniques such as centrifugation or cell sorting.
  • cell-, tissue- or organ samples are obtained from tumor tissues.
  • the sample is a sample comprising tumor cells, more preferably a tumor sample, preferably a formalin- fixed tumor sample, more preferably a formalin- fixed, paraffin embedded tumor sample.
  • the sample is a sample comprising colorectal cancer cells, more preferably a tumor sample of a colorectal cancer, preferably a formalin- fixed tumor sample of a colorectal cancer, more preferably a formalin- fixed, paraffin embedded tumor sample of a colorectal cancer.
  • colonal cancer is, in principle, known to the skilled person as relating to a cancer originating in the colon (colon cancer) or in the rectum (rectal cancer).
  • metastases of other cancers having the primary tumor in other parts of the body are not colorectal cancer, even if said metastases are situated in the colon or rectum.
  • the colorectal cancer is an adenocarcinoma, a carcinoid tumor, a gastrointestinal stromal tumor, a lymphoma, or a sarcoma. More preferably, the colorectal cancer is an adenocarcinoma.
  • colorectal cancer may be of any of cancer stages I to IV.
  • CpG and CpG site are known to the skilled person.
  • the terms relate to a site in DNA, preferably chromosomal DNA of a subject, having the nucleotide sequence 5'-CG-3'.
  • CpG sites can be methylated by DNA methyltransferases at the cytosine residue to yield a 5-methylcytosine residue, and methylation at a specific CpG site may be inherited or may be a de novo methylation acquired during life time of the subject.
  • the CpG sites as referred to herein are those of Table 1.
  • the CpG site locations indicated in Table 1 refer to the positions in the human reference genome GRCh37 as provided by the Genome Reference Consortium (www.ncbi.nlm.nih.gov/grc) on 2009/02/27. This assembly is also referred to as hg 19.
  • CpG site related to a CpG site X, with X being a specific CpG site of Table 1, as used herein, relates to a CpG site in the vicinity of the specific CpG site.
  • a CpG site related to a specific CpG site is a CpG site at most 5 kb downstream or upstream, more preferably at most 2 kb downstream or upstream, even more preferably at most 1 kb downstream or upstream, still more preferably 0.5 kb downstream or upstream of the specific CpG site.
  • the CpG site related to a specific CpG site is the specific CpG site of Table 1 itself.
  • a CpG site related to a CpG site of Table 1 is a CpG site of Table 1 as specified herein above, i.e. is a CpG site specifically indicated in Table 1.
  • the present invention preferably relates to a method for determining a survival probability of a subject suffering from colorectal cancer comprising a) detecting the methylation status of at least two CpG sites selected from the CpG sites of Table 1 in a sample of said subject and,
  • step b) based on the methylation status detected in step a), determining the survival probability of said subject.
  • the term "at least two CpG sites related to at least two CpG sites of Table 1", as used herein, relates to at least to CpG sites related to two non-identical CpG sites of Table 1.
  • more than one CpG site related to the same CpG site of Table 1 may preferably be analyzed; e.g. in case three CpG sites are analyzed, one CpG site related to eg 16336556 and two CpG sites related to eg 14270346 might be analyzed. More preferably, however, the number of CpG sites analyzed is identical to the number of non- identical CpG sites of Table 1 these CpG sites analyzed are related to.
  • Table 1 CpG sites of the invention; positions on human chromosome and nucleotide number of the CpG sites refer to the human genome sequence assembly GRCh37/hgl9.
  • the CpG sites analyzed according to the method of the present invention comprise CpG sites related to CpG sites selected from the list consisting of cgl6336556, cgl4270346, cg05646575, cgl7431888, cgl2510999, cg00832644, cgl 1056055, cg08804626, cgl4983135, cg22522598, cgl9184885, cg08729279, and cgl0758824; preferably consisting of cgl6336556, cgl4270346, cg05646575, cgl7431888, cgl2510999, cg00832644, and cgl 1056055.
  • the CpG sites analyzed according to the method of the present invention comprise CpG sites selected from the list consisting of cgl6336556, cgl4270346, cg05646575, cgl7431888, cgl2510999, cg00832644, cgl 1056055, cg08804626, cgl4983135, cg22522598, cgl9184885, cg08729279, and cgl0758824; preferably consisting of cgl6336556, cgl4270346, cg05646575, cgl7431888, cgl2510999, cg00832644, and cgl 1056055.
  • methylation status relates to a state of a specific CpG site in a cell being methylated or not, more preferably relates to the extent to which a specific CpG site is methylated in a population of cells, or not.
  • a specific CpG site there are four occurrences of a specific CpG site, i.e. two alleles, with each allele comprising the two strands of DNA making up double-stranded DNA; thus, the methylation status of a single CpG site may be all four CpGs non-methylated; one CpG methylated; two, three, or four CpGs methylated.
  • the methylation of only one strand of a given DNA is analyzed, e.g. by hybridizing a primer upstream of said CpG site as specified herein below.
  • the methylation status of a CpG site is not necessarily identical for all cells of said population.
  • the methylation status is detected as the number of cells comprising a specific CpG site at least one, preferably at least twice, in methylated form in a given number of cells; or the methylation status is detected as the number of methylated forms of a specific CpG site detected in a given number of cells.
  • beta-values range from 0 to 1, with 0 representing completely unmethylated and 1 represents completely methylated.
  • the methylation status of a CpG site in a population of cells preferably, is the average degree of methylation of said CpG site in a population of at least 10, preferably at least 25, more preferably at least 100 cells.
  • the methylation status may also be expressed as a ratio of the number of individual CpG sites at a given position found to be unmethylated to the total number of individual CpG sites at said given position analyzed, i.e. as a non-methylation status. More preferably, the methylation status is expressed as a ratio of the number of individual CpG sites at a given position found to be methylated to the total number of individual CpG sites at said given position analyzed.
  • the method comprises isolating genomic DNA from said sample, preferably from cells comprised in said sample.
  • the method comprises contacting said DNA with a methylation-sensitive restriction enzyme having a nucleic acid sequence comprising the sequence 5'-CG-3' as a recognition sequence; preferably, the method further comprises contacting a further aliquot of said DNA with a corresponding non-methylation-sensitive restriction enzyme having the same nucleic acid sequence comprising the sequence 5'-CG-3' as a recognition sequence.
  • the method comprises treating said DNA, before or after isolation, with a bisulfite, preferably sodium bisulfite.
  • the method further comprises annealing an oligonucleotide specifically annealing to a sequence immediately upstream of said CpG site and comprising a 3'-terminal sequence 5'-CG-3' and/or an oligonucleotide specifically annealing to a sequence immediately upstream of said CpG site and comprising a 3 '-terminal sequence 5'-CA-3' to said genomic DNA, preferably to said bisulfite-treated genomic DNA, per CpG site.
  • the method further comprises performing a one-nucleotide extension reaction after said annealing in such case.
  • the method comprises annealing per CpG site an oligonucleotide specifically annealing to a sequence immediately upstream of said CpG site and having a C as the terminal nucleotide, and performing pyrosequencing using said oligonucleotide as a sequencing primer.
  • a sequencing primer e.g., a sequencing primer for determining whether the oligonucleotide is a sequence immediately upstream of said CpG site and having a C as the terminal nucleotide.
  • methylation of only the strand complementary to said oligonucleotide is analyzed.
  • methylation of the CpG site of only one strand of DNA is analyzed, namely the CpG site as indicated above in Table 1 ; thus, preferably, for each CpG site indicated in Table 1, only one oligonucleotide is used in analysis.
  • the methylation status of at least two CpG sites selected from Table 1 is determined.
  • accuracy of prediction may be increased by determining the methylation status of an increased number of CpG sites; thus, preferably, the methylation status of at least three, preferably at least five, more preferably at least eight, most preferably at least 13 of said CpG sites of Table 1 is determined.
  • the methylation status of at least three, preferably at least five, more preferably at least six CpG sites related to, more preferably CpG sites selected from cgl6336556, cgl4270346, cg05646575, cgl7431888, cgl2510999, cg00832644, and cgl 1056055 is detected.
  • the methylation status of all methylation sites of Table 1 is determined.
  • a survival probability is determined by comparing the methylation status determined in a sample to a corresponding reference.
  • the method for determining a survival probability comprises comparing the methylation status determined for a CpG site in a sample to a reference.
  • the method comprises further step al) comparing the methylation status of said at least two CpG sites of step a) to references; and in step b) the determining is based on the comparison of step al).
  • the term "reference” relates to a reference value or a reference range, preferably derived from a population of subjects, preferably a population of apparently healthy subjects as specified herein above, more preferably a population of subjects suffering from colorectal carcinoma as specified elsewhere herein.
  • a reference value or reference range may be obtained from a second sample, which is a sample of healthy tissue, more preferably of healthy tissue of the same tissue type, more preferably of healthy tissue from the same subject, most preferably of the same tissue type and from the same subject as the tumor sample.
  • the term “corresponding reference” relates to a reference value or reference range obtained by applying the same method, but to a different, i.e. reference, sample.
  • determining the methylation status of cgl6336556 in a sample of an apparently healthy subject and/or determining the methylation status of cgl6336556 in a sample of healthy tissue of the same patient provides a preferred corresponding reference.
  • a score is calculated e.g. from a multitude of CpG sites, calculating a corresponding score from the same CpG sites of a sample of an apparently healthy subject and/or a sample of healthy tissue of the same patient provides a preferred corresponding reference .As indicated above, at least two CpG sites are evaluated according to the present invention.
  • the value detected for a specific CpG site is compared to a reference for a corresponding CpG site, i.e. to a reference value or reference range pertaining to the CpG site having the same position in the genome.
  • a reference value or reference range pertaining to the CpG site having the same position in the genome e.g. the average degree of methylation is determined for the eight first CpG sites of Table 1, each of these values is compared to a corresponding reference value, respectively.
  • values are compared to corresponding values, i.e. average degree of methylation values are compared to average degree of methylation values, numbers of cells comprising the CpG site in methylated form are compared to numbers of cells comprising the CpG site in methylated form, and the like.
  • the above applies mutatis mutandis.
  • the reference is derived from healthy tissue of the same patient, from a population of apparently healthy subjects. More preferably, the reference includes a value of a methylation status or a score derived therefrom representing an average value of a population of subjects suffering colorectal cancer; more preferably the reference is a median of a methylation status or a score derived therefrom of a population of subjects suffering colorectal cancer.
  • an unfavorable health state is determined if at least one of said CpG sites deviates from, preferably significantly deviates from, more preferably is lower than, most preferably is significantly lower than, the reference value.
  • an unfavorable survival probability is determined if a methylation status deviating from, preferably significantly deviating from, more preferably being lower than, most preferably significantly lower than, the reference is detected for at least two, more preferably at least four, even more preferably at least six, still more preferably at least eight, most preferably more than ten CpG sites.
  • an unfavorable survival probability is determined if a methylation status deviating from, preferably significantly deviating from, more preferably being lower than, most preferably being significantly lower than, the reference is detected for at least two, more preferably at least four, even more preferably at least six, still more preferably at least eight, most preferably at least ten CpG sites selected from the CpG sites of Table 1, preferably selected from the first 13 CpG sites of Table 1.
  • a decrease of the methylation status of a CpG site compared to the reference is indicative of an unfavorable survival probability; and a non-decreased methylation status, i.e. preferably being similar or increased compared to the reference is indicative of a favorable survival probability.
  • a multitude of CpG sites is analyzed and the respective methylation statuses are combined into a score.
  • Said score may be obtained by, e.g. summing up the ⁇ values of the respective CpG sites; preferably, said sum is compared to the corresponding sum obtained from a reference.
  • the ⁇ values are weighted for obtaining a score, more preferably with the weighting factors of Table 1 ("PCA weight");
  • PCA weight weight
  • weighting factors may be used; preferably, however, these weighting factors for all CpG sites used in the calculation are derived from the aforesaid weighting factors by a common mathematical operation, more preferably multiplication or division by the same factor.
  • the score calculated according to eq. 1 may assume values of from 0 (all CpG sites having a ⁇ value of 0, i.e. being unmethylated) to 4.30 (all CpG sites having a ⁇ value of 1, i.e. being fully methylated).
  • a methylation status increased compared to the median methylation status of a population of subjects suffering from colorectal cancer is indicative of a favorable survival probability; also preferably, a methylation status decreased compared to the median methylation status of a population of subjects suffering from colorectal cancer is indicative of an unfavorable survival probability.
  • a score, preferably calculated according to eq. 1, increased compared to the median score of a population of subjects suffering from colorectal cancer is indicative of a favorable survival probability; also preferably, a score, preferably calculated according to eq. 1, decreased compared to the score of a population of subjects suffering from colorectal cancer is indicative of an unfavorable survival probability.
  • a score calculated according to eq. 1 being higher than about 2.85, more preferably higher than 2.86, is indicative of a favorable survival probability; also preferably, a score being lower than about 2.85, more preferably lower than or equal to 2.86, is indicative of an unfavorable survival probability.
  • a value and a reference value are determined to be essentially identical if the difference between two values is, preferably, not significant and shall be characterized in that the value is within at least the interval between 1st and 99th percentile, 5th and 95th percentile, 10th and 90th percentile, 20th and 80th percentile, 30th and 70th percentile, 40th and 60th percentile of the reference value, preferably, the 50th, 60th, 70th, 80th, 90th or 95th percentile of the reference value.
  • an observed difference for two values shall preferably be statistically significant.
  • a difference in value is, preferably, significant outside of the interval between 45th and 55th percentile, 40th and 60th percentile, 30th and 70th percentile, 20th and 80th percentile, 10th and 90th percentile, 5th and 95th percentile, 1st and 99th percentile of the reference value.
  • Whether a difference is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools, e.g., determination of confidence intervals, p-value determination, Student ' s t-test, Mann- Whitney test etc.. Details are found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983.
  • Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99 %.
  • the p-values are preferably ⁇ 0.1 , more preferably ⁇ 0.05, still more preferably ⁇ 0.01, even more preferably ⁇ 0.005, or, most preferably ⁇ 0.0001.
  • the probability envisaged by the present invention allows that the determination will be correct for at least 60%, more preferably at least 70%, still more preferably at least 80%>, or, most preferably, at least 90%> of the subjects of a given cohort or population. Further methods of evaluating statistical significance of differences in methylation are described herein below in the Examples.
  • a reference value may be established by determining the mean methylation status of a population of subjects suffering from colorectal cancer known to have an unfavorable survival probability, and may be used as a cutoff value; as will be understood, in such case a methylation status similar to the reference would be indicative of an unfavorable survival probability.
  • the lower limit of normal (LLN) may be used as a cutoff.
  • values derived from any of the aforesaid populations may be divided in halves, tertiles, quartiles, pentiles, or the like.
  • the lowest or the lower two tertiles of values of a population of subjects suffering from colorectal cancer may be regarded as being indicative of unfavorable survival probability.
  • the specific choice of reference and/or score will mainly be governed on the specific sensitivity and specificity required, but also by other parameters such as the particular population of interest.
  • the skilled person has means and methods at hand enabling appropriate election.
  • the methylation status of the indicated CpG sites is an independent indicator of the mortality risk of a subject suffering from colorectal cancer.
  • a prognostic classifier preferably based on 20 CpGs, could be constructed, which improves prognosis prediction for overall survival and disease-free survival colorectal cancer patients and which provides for a significant reduction of prediction error.
  • the definitions made above apply mutatis mutandis to the following. Additional definitions and explanations made further below also apply for all embodiments described in this specification mutatis mutandis.
  • the present invention further relates to a method for treating colorectal cancer in a subject suffering from colorectal cancer comprising
  • the method of treating a subject of the present invention preferably, is an in vivo method. Moreover, it may comprise steps in addition to those explicitly mentioned above. For example, further steps may relate, e.g., to diagnosing colorectal cancer before the steps of the method for determining a survival probability, providing further therapeutic options, or administering one or more therapeutic measures to said subject, depending on the result of said method. Moreover, one or more of said steps may be performed by automated equipment.
  • the possibility to establish a survival probability for a subject enables the medical practitioner to better select an appropriate therapy.
  • the primary tumor is removed by surgery, including classical surgery, i.e. resection of tumor, ablation (e.g. radio frequency ablation), and cryotherapy (cryosurgery).
  • ablation e.g. radio frequency ablation
  • cryotherapy cryosurgery
  • the method of treating a subject of the present invention preferably, comprises the step of removal of the primary tumor, more preferably before or after further treatment is administered.
  • the method of treating a subject further comprises providing at least one of close monitoring and/or lifestyle recommendations and/or treatment methods to said subject.
  • close monitoring relates to medically examining a subject for signs of relapse and/or metastasis at least once within 3 months, preferably within two months, more preferably within one month for a period of at least 12 months, more preferably at least 18 months, still more preferably at least 24 months, most preferably at least 35 months.
  • lifestyle recommendations as used herein, relates to recommendations decreasing the probability of relapse and/or metastasis. Preferably, such recommendations are recommendations to reduce or quit alcohol consumption, to reduce or quit smoking, to reduce body weight, to increase exercise and/or to use healthy nutrition.
  • chemotherapy relates to treatment of a subject with an antineoplastic drug.
  • chemotherapy is a treatment including alkylating agents (e.g. cyclophosphamide), platinum (e.g. carboplatin), antimetabolites (e.g. 5-Fluorouracil), anthracyclines (e.g. doxorubicin, epirubicin, idarubicin, or daunorubicin), topoisomerase II inhibitors (e.g.
  • etoposide etoposide, irinotecan, topotecan, camptothecin, or VP 16
  • anaplastic lymphoma kinase (ALK)-inhibitors e.g. Crizotinib or AP26130
  • aurora kinase inhibitors e.g.
  • chemotherapy preferably, relates to a complete cycle of treatment, i.e. a series of several (e.g. four, six, or eight) doses of antineoplastic drug or drugs applied to a subject, which may be separated by several days or weeks without such application.
  • radiation therapy and “radiotherapy” are known to the skilled artisan.
  • the term relates to the use of ionizing radiation to treat or control cancer.
  • targeted therapy relates to application to a patient a chemical substance known to block growth of cancer cells by interfering with specific molecules known to be necessary for tumorigenesis or cancer or cancer cell growth.
  • Examples known to the skilled artisan are small molecules like, e.g. PARP-inhibitors (e.g. Iniparib), antiangiogenic agents (e.g. Bevacizumab, Ramucirumab, Ziv-aflibercept), signalling inhibitors (e.g. cetuximab or panitumumab), or kinase inhibitors (e.g. Regorafenib).
  • PARP-inhibitors e.g. Iniparib
  • antiangiogenic agents e.g. Bevacizumab, Ramucirumab, Ziv-aflibercept
  • signalling inhibitors e.g. cetuximab or panitumumab
  • kinase inhibitors e.g. Regorafenib
  • immunotherapy as used herein relates to the treatment of cancer by modulation of the immune response of a subject. Said modulation may be inducing, enhancing, or suppressing said immune response, e.g. by administration of at least one cytokine, and/or of at least one antibody specifically recognizing cancer cells.
  • cell based immunotherapy relates to a cancer therapy comprising application of immune cells, e.g. T- cells, preferably tumor-specific NK cells, to a subject.
  • the present invention also relates to a method for patient monitoring comprising the steps of the method for determining a survival probability and/or of the method for treating colorectal cancer of the present invention and the further steps or steps of providing close monitoring and/or lifestyle recommendations in case an unfavorable survival probability and/or an increased mortality risk is detected.
  • the present invention further relates to a use of means for determining the methylation status of, at least two CpG sites related to at least two CpG sites of Table 1, preferably at least two CpG sites of Table 1, for manufacturing a diagnostic means or device for determining a survival probability of a subject suffering from colorectal cancer.
  • the present invention also relates to a data collection, preferably comprised on a data carrier, comprising the positions of at least two, preferably at least three, more preferably at least five, even more preferably at least eight, still more preferably at least 13, most preferably at least 16 CpG sites selected from Table 1 and/or CpG sites related thereto; preferably of from three to all, more preferably of from ten to all, even more preferably of froml5 to all CpG sites selected from Table 1 and/or CpG sites related thereto.
  • the data collection further comprises reference values or reference ranges for the methylation status of said CpG sites, preferably as specified elsewhere herein, more preferably as specified herein in the Examples.
  • data collection refers to a collection of data which may be physically and/or logically grouped together. Accordingly, the data collection may be implemented in a single data storage medium or in physically separated data storage media being operatively linked to each other.
  • the data collection is implemented by means of a database.
  • a database as used herein comprises the data collection on a suitable storage medium.
  • the database preferably, further comprises a database management system.
  • the database management system is, preferably, a network-based, hierarchical or object-oriented database management system.
  • the database may be a federal or integrated database. More preferably, the database will be implemented as a distributed (federal) system, e.g. as a Client-Server-System.
  • the database is structured as to allow a search algorithm to compare a test data set with the data sets comprised by the data collection. Specifically, by using such an algorithm, the database can be searched for similar or identical data sets being indicative for a survival probability as set forth above (e.g. a query search). Consequently, the information obtained from the data collection can be used, e.g., as a reference for the methods of the present invention described above.
  • the term "data storage medium” as used herein encompasses data storage media which are based on single physical entities such as a CD, a CD-ROM, a hard disk, optical storage media, a diskette, or a sheet of paper. Moreover, the term further includes data storage media consisting of physically separated entities which are operatively linked to each other in a manner as to provide the aforementioned data collection, preferably, in a suitable way for a query search.
  • the present invention relates to a kit comprising means for determining the methylation status of at least two CpG sites related to at least two CpG sites of Table 1 , preferably at least two CpG sites selected from the CpG sites of Table 1, more preferably selected from the list consisting of cgl6336556, cgl4270346, cg05646575, cgl7431888, cgl2510999, cg00832644, cgl 1056055, cg08804626, cgl4983135, cg22522598, cgl9184885, cg08729279, and cgl0758824 in a sample of a subject suffering from colorectal cancer, and a data collection according to the present invention.
  • the present invention relates to a device comprising an analysis unit comprising means for determining the methylation status of at least two CpG sites related to at least two CpG sites of Table 1, preferably of at least two CpG sites selected from the CpG sites of Table 1, more preferably selected from the list consisting of in a sample of a subject suffering from colorectal cancer, and an evaluation unit comprising a data collection according to the present invention.
  • a method for determining a survival probability of a subject suffering from colorectal cancer comprising a) detecting the methylation status of at least two CpG sites related to at least two CpG sites of Table 1 in a sample of said subject and,
  • step b) based on the methylation status detected in step a), determining the survival probability of said subject.
  • determining said survival probability comprises determining a mortality risk.
  • any one of embodiments 1 to 17, wherein said method comprises annealing an oligonucleotide specifically annealing to a sequence immediately upstream of said CpG site and comprising a 3'-terminal sequence 5'-CG-3' and/or an oligonucleotide specifically annealing to a sequence immediately upstream of said CpG site and comprising a 3 '-terminal sequence 5 -CA-3' to said genomic DNA, preferably to said bisulfite-treated genomic DNA, per CpG site.
  • a method for treating colorectal cancer in a subject suffering from colorectal cancer comprising
  • a method for patient monitoring comprising the steps of the method according to any one of embodiments 1 to 24 and providing close monitoring and/or lifestyle recommendations in case an unfavorable survival probability and/or an increased mortality risk is detected.
  • a data collection preferably comprised on a data carrier, comprising the positions of at least two, preferably at least three, more preferably at least five, even more preferably at least eight, most preferably at least 15 CpG sites selected from Table 1; preferably of from three to all, more preferably of from ten to all, even more preferably of froml5 to all CpG sites selected from Table 1 and/or CpG sites related thereto.
  • a kit comprising means for determining the methylation status of at least two CpG sites related to at least two CpG sites of Table 1 , preferably at least two CpG sites selected from the CpG sites of Table 1, more preferably selected from the list consisting of cgl6336556, cgl4270346, cg05646575, cgl7431888, cgl2510999, cg00832644, cgl 1056055, cg08804626, cgl4983135, cg22522598, cgl9184885, cg08729279, and eg 10758824 in a sample of a subject suffering from colorectal cancer, and a data collection according to embodiment 29 or 30.
  • a device comprising an analysis unit comprising means for determining the methylation status of at least two CpG sites related to at least two CpG sites of Table 1 , preferably of at least two CpG sites selected from the CpG sites of Table 1, more preferably selected from the list consisting of in a sample of a subject suffering from colorectal cancer, and an evaluation unit comprising a data collection according to embodiment 29 or 30.
  • Fig. 1 Study design and analysis flowchart
  • Fig. 2 Illustration of weighting in the IHW model.
  • the left side of the plot, with ⁇ -value difference (x-axis) ranging between -0.5 and 0, represents the CpG sites with lower methylation in tumor than in normal mucosa tissue
  • the right side with ⁇ -value difference between 0 and 0.5 represents the CpG sites with higher methylation in tumor than in normal mucosa tissue.
  • the highest weighting (y-axis) was given to CpG sites with clearly lower methylation levels in tumor than in normal mucosa tissue and no weight was given to CpG sites with higher methylation levels in tumor tissue.
  • Fig. 3 Prediction error curve and AUC curve for OS (A, B) and DSS (C, D) of CRC patients with non-metastatic disease.
  • the prediction error was calculated based on the clinical variables age, gender, smoking behavior, MSI-status, tumor stage and tumor location (colon proximal, colon distal, rectum).
  • the prediction error was further reduced.
  • Fig. 4 Kaplan-Meier plots for OS of all CRC patients with non-metastatic disease (stage I-III) dependent on the methylation status of the combined methylation marker analyzed in the screening cohort (A) and in the validation cohort (B).
  • OS of only stage II and III CRC patients dependent on the methylation status of the combined methylation marker is shown for the screening cohort (C) and the validation cohort (D).
  • Group low low expression of the combined methylation marker (value ⁇ median)
  • group high high expression of the combined methylation (value > median). Given p-values are based on log-rank tests between the respective groups.
  • the shape of the Kaplan-Meier plots support the assumption of proportional hazards.
  • the following Examples shall merely illustrate the invention. They shall not be construed, whatsoever, to limit the scope of the invention.
  • Example 1 Study cohort
  • the validation cohort contained corresponding normal mucosa tissue samples, enabling the analysis of tumor and normal mucosa tissue pairs for 34 patients. Patients who had received neoadjuvant therapy were excluded for the survival analysis. The median FU-time for all patients in the screening and the validation cohort was approximately five (4.99) years. Table 2 gives an overview of the clinicopathological characteristics of the included patients.
  • Tissue samples were collected from all de-central pathology institutes in the tissue bank of the National Center for Tumor Diseases (NCT, Heidelberg, Germany). All samples were provided in accordance with the regulations of the tissue bank and the approval of the ethics committee of Heidelberg University. For each sample, tumor DNA was isolated from four formalin- fixed and paraffin-embedded (FFPE) tissue slices a 5 ⁇ . A hematoxylin-stained slice of every tumor block was evaluated by an experienced pathologist in order to mark the regions with high tumor cell content.
  • FFPE formalin- fixed and paraffin-embedded
  • the DNA of the manually microdissected tumor tissue of the screening cohort was isolated following a semi-automated protocol using the Maxwell® 16 MDx instrument (Promega, USA) in combination with the DNeasy Blood & Tissue Kit (Qiagen, Germany) whereas the DNA of the validation cohort was extracted manually with the DNeasy Blood & Tissue Kit according to manufacturer's recommendations.
  • the isolated DNA was eluted with 50 ⁇ 1 elution buffer.
  • Example 3 Methylation profiling using the Infmium HumanMethylation450 BeadChip array (Illumina)
  • Preprocessing and statistical analyses were all performed using the computational environment R, version 3.3.1 (www.r-project.org/).
  • Raw data files generated by the iScan array scanner were read and preprocessed using the 'minfi' package, included in the Bioconductor collection of R packages.
  • the standard Illumina normalization procedure ('preprocesslllumina') was used to correct for technical differences between the Infinium I and II assay designs.
  • filtering criteria were applied according to Sturm et al. (Cancer cell 2012;22(4):425-37). In the screening cohort, probes that failed in more than 10% samples, based on detection p-value using a significance level of 0.01, were excluded. To allow for an independent validation, no filtering was applied to the validation cohort.
  • a paired Wilcoxon signed-rank test was used to find the differentially methylated CpG sites between the tumor and normal tissue pairs. The difference of tumor and normal tissues for every CpG site was estimated via the (pseudo-)median of the sample differences. For statistical inference, a filtering step for standard deviation > 0.05 was applied to the selected CpG sites.
  • a variable screening for prognostic CpG sites for overall survival (OS) was performed for single CpG sites using marginal testing based on a Cox model adjusted for age as continuous covariate and gender, smoking behavior, MSI-status, tumor stage (2 vs. 1, 3 vs. 1) and tumor location (colon proximal, colon distal or rectum) as categorical covariates.
  • IHW independent hypothesis weighting
  • the first model included only the clinical covariates (as specified above), the second one included both the clinical covariates and the prognostic classifier.
  • the given hazard ratios (HR) are based on a change from the lower to the upper quartile of the prognostic classifier.
  • the Cox model based only on clinical covariates and the Cox model based on clinical covariates and the prognostic classifier were independently validated in the validation cohort.
  • the prediction error curve and the Brier score for the 3 -years survival in the validation cohort were evaluated using the loss function approach described in Gerds and Schumacher (Biometrical Journal 2006;48(6): 1029-40) using the R package 'pec'.
  • the area under the curve (AUC) curves were calculated following the incident/dynamic approach described in the work of Song and Zhou (Statistica Sinica 2008(18):947-65) using the R package 'survAUC.
  • Example 6 Analysis of CpG based methylation data from the genome-wide analysis
  • Example 7 Identification of candidate CpG sites for the OS of patients with non-metastatic CRC
  • the Brier Score a measure for the accuracy of probabilistic predictions, was calculated for the 3-years survival of the patients with non-metastatic disease.
  • the 20 CpG sites with the best Brier Score were selected for the prognostic classifier.
  • ProMCol classifier Using the ProMCol classifier, we fitted a Cox regression analysis in the screening cohort. In one model only the standard clinical variables (gender, age, tumor stage, tumor location, smoking behavior and MSI-status) were included, in the other model the clinical variables in combination with the ProMCol classifier.
  • the prediction error for the OS of patients with non- metastatic CRC was calculated in the validation cohort.
  • This analysis revealed a clear advantage in prediction probability for adding the ProMCol classifier to the model ( Figure 3A).
  • the prediction error, calculated by the model using only clinical variables was 0.127 for the three-year survival of the patients
  • a model combining the clinical variables with the ProMCol classifier improved the prediction with a smaller error value of 0.120.
  • the prediction error could be reduced from 0.153 with clinical variables to 0.140 using the combination of clinical variables and the ProMCol classifier.
  • Cancer stage (2 vs. 1) 1.45 0.84-2.51 0.181 1.24 0.71-2.15 0.451

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Abstract

La présente invention concerne un procédé de détermination d'une probabilité de survie d'un sujet souffrant d'un cancer colorectal comprenant a) la détection de l'état de méthylation d'au moins deux sites CpG liés à au moins deux sites CpG sélectionnés parmi cg16336556, cg14270346, cg05646575, cg17431888, cg12510999, cg00832644, cg11056055, cg08804626, cg14983135, cg22522598, cg19184885, cg08729279, cg10758824, cg18195165, cg08617020, cg23750514, cg01131395, cg18736676, cg19340296, et cg16399624 dans un échantillon dudit sujet et, b) sur la base de l'état de méthylation détecté dans l'étape a), la détermination de la probabilité de survie dudit sujet ; et un procédé de suivi du patient comprenant les étapes ci-dessus mentionnées et fournissant un suivi étroit et/ou des recommandations de style de vie dans le cas de probabilité défavorable de survie et/ou dans lequel un risque accru de mortalité est détecté. La présente invention concerne en outre une collecte de données comprenant les positions d'au moins deux des sites CpG de l'invention et des kits, des dispositifs, et leurs utilisations.
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