WO2019081507A1 - Nouveaux marqueurs dérivés du sang pour la détection du cancer - Google Patents

Nouveaux marqueurs dérivés du sang pour la détection du cancer

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Publication number
WO2019081507A1
WO2019081507A1 PCT/EP2018/079035 EP2018079035W WO2019081507A1 WO 2019081507 A1 WO2019081507 A1 WO 2019081507A1 EP 2018079035 W EP2018079035 W EP 2018079035W WO 2019081507 A1 WO2019081507 A1 WO 2019081507A1
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WIPO (PCT)
Prior art keywords
cancer
cpg
mir
methylation
seq
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PCT/EP2018/079035
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English (en)
Inventor
Barbara BURWINKEL
Xue CAO
Quiqiong TANG
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Universität Heidelberg
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Publication of WO2019081507A1 publication Critical patent/WO2019081507A1/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
    • 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
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the present invention relates to the field of pharmacogenomics and in particular to detecting the presence of CpG methylation and miRNAs in blood for the detection of cancer. This detection is useful for a minimally invasive diagnosis of cancer as well as for monitoring cancer treatment and assessing treatment response.
  • the invention provides methods suitable for this purpose.
  • DNA methylation is a type of epigenetic alteration which plays an important role in cancer development. Promoter hypermethylation of tumor suppressor genes and global hypomethylation leading to malignancy have been studied extensively in different cancer types. Global DNA hypomethylation is a hallmark of most cancers, including breast cancer. This DNA hypomethylation has been proposed to activate oncogenes, induce genomic instability and promote choromosome instability. Genome-wide DNA hypomethylation originates from the decrease of 5-methyldeoxycytosine (5-mdC) in dinucleotide CpG sites throughout the genome. As most 5- mdC sites are rich in repetitive sequences that account for approximately half of the human genome, and those repetitive DNA sequences are highly methylated in normal cells.
  • 5-mdC 5-methyldeoxycytosine
  • repetitive sequences dispersed throughout the genome, such as long interspersed nuclear elements, short interspersed nuclear elements and satellite repeats.
  • LINEl a long interspersed nuclear element, is scattered throughout about 17% of the entire genome.
  • Alu is a short interspersed repetitive sequence that contributes almost 11% of the human genome.
  • the DNA methylation of repetitive elements has been associated with global DNA methylation and used as a marker for global methylation status by some investigators.
  • global hypomethylation in peripheral blood DNA has been considered as a risk factor for many tumors, such as colorectal, bladder as well as head and neck cancer.
  • MiRNAs are small, non-coding RNAs (-18-25 nucleotides in length) that regulate gene expression on a post-transcriptional level by degrading mRNA molecules or blocking their translation. Hence, they play an essential role in the regulation of a large number of biological processes, including cancer. MiRNAs can be found in body fluids like blood. Such circulating miRNAs have been reported as aberrantly expressed in blood plasma or serum in different types of cancer, e.g. prostate, colorectal or esophageal carcinoma. They are relatively stable and can be measured repeatedly in a minimally invasive manner.
  • LINEl and Alu methylation as well as several miRNAs can be used as blood markers for the detection of cancer, in particular of breast, ovarian and pancreatic cancer.
  • the present invention relates to a method for detecting the presence or absence of cancer in a subject, comprising the step of determining the level of cytosine methylation of at least one CpG dinucleotide selected from the group consisting of LINEl CpG dinucleotides 1, 3, 4, 5, 9, 12, and 14 of SEQ ID NO: 1, CpG dinucleotides within SEQ ID NO: 3 that are co- methylated with LINEl CpG dinucleotide 1 of SEQ ID NO: l, and Alu CpG dinucleotides 13 and 14 of SEQ ID NO: 2 in genomic DNA from a sample of the subject, wherein hypomethylation is indicative of the presence of the cancer and the lack thereof is indicative of the absence of the cancer.
  • the present invention relates to a method for diagnosing cancer or for screening for cancer, comprising detecting the cancer according to the first aspect.
  • the present invention relates to a method for monitoring a subject having an increased risk of developing cancer, comprising detecting the cancer according to the first aspect repeatedly.
  • the present invention relates to a method for monitoring cancer treatment of a subject, comprising detecting the cancer according to the first aspect repeatedly across the treatment period.
  • the present invention relates to a method for assessing the response of a subject to a cancer treatment, comprising detecting the cancer according to the first aspect during and/or after the treatment.
  • the present invention relates to a method for treating a subject having cancer detected according to the method of the first aspect, comprising administering a cancer therapy to the subject.
  • Figure 1 Box plot of LINE1.
  • Figure 2 ROC curve for LINEl_CpG_l methylation (A) and LINE1 mean methylation (B).
  • Figure 3 Box plot of Alu_CpG_13 and Alu_CpG_14.
  • FIG. 4 ROC curve for Alu_CpG_13 methylation (A) and Alu_CpG_14 methylation (B). ROC curve was calculated by logistic regression.
  • Figure 6 ROC curve for LINEl_CpG_l and Alu_CpG_13.
  • the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, H.G.W, Nagel, B. and Kolbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).
  • the present invention relates to a method for detecting the presence or absence of cancer in a subject, comprising the step of determining the level of cytosine methylation of at least one CpG dinucleotide selected from the group consisting of LINE1 CpG dinucleotides 1, 3, 4, 5, 9, 12, and 14 of SEQ ID NO: 1, CpG dinucleotides within SEQ ID NO: 3 that are co- methylated with LINE1 CpG dinucleotide 1 of SEQ ID NO: l, and Alu CpG dinucleotides 13 and 14 of SEQ ID NO: 2 in genomic DNA from a sample of the subject, wherein hypomethylation is indicative of the presence of the cancer and the lack thereof is indicative of the absence of the cancer.
  • CpG dinucleotides can be used as univariate markers or as multivariate markers.
  • the level of cytosine methylation of Alu CpG dinucleotide 13 of SEQ ID NO: 2 is determined.
  • the inventors have shown that this is one of the most distinctive CpG sites and useful in particular as a univariate marker.
  • the level of cytosine methylation of LINEl CpG dinucleotide 1 of SEQ ID NO: 1 is determined. The inventors have shown that this is the most distinctive CpG site and useful as a univariate marker and a multivariate marker irrespective of other markers used in combination.
  • CpG dinucleotide selected from the group consisting of LINEl CpG dinucleotides 1, 3, 4, 5, 9, 12, and 14 of SEQ ID NO: 1 and/or of at least one (including two) CpG dinucleotide selected from the group consisting of Alu CpG dinucleotides 13 and 14 of SEQ ID NO: 2.
  • the above methods of the first aspect determining the methlyation level of one or more LINEl CpG dinucleotides further comprise determining the level of cytosine methylation of at least one (including at least two, at least three or of four) further CpG dinucleotide selected from the group consisting of LINEl CpG sites 2, 15, 16 and 17 of SEQ ID NO: 1.
  • the above methods of the first aspect determining the methlyation level of one or more Alu CpG dinucleotides further comprise determining the level of cytosine methylation of at least one (including at least two, at least three or of four) further CpG dinucleotide selected from the group consisting of Alu CpG dinucleotides 1, 2, 11 and 12 of SEQ ID NO: 2.
  • the above methods of the first aspect comprising determining the methlyation level of both one or more LINEl and one or more Alu CpG dinucleotides comprise determining the level of cytosine methylation of LINEl CpG dinucleotides 1, 2, 14, 16 and 17 of SEQ ID NO: 1 and of Alu CpG dinucleotides 1, 2, 11, 12 and 14 of SEQ ID NO: 2.
  • the methylation level of one of LINEl CpG dinucleotides 3, 4 or 5 is determined, the methylation level of one or two of the remaining two is also determined, i.e. of 3 and 4, 3 and 5, 4 and 5 or 3 to 5;
  • the methylation level of one of LINEl CpG dinucleotides 16 or 17 is determined, the methylation level of the other one is also determined, i.e. of 16 and 17;
  • the methylation level of one of Alu CpG dinucleotides 11 or 12 is determined, the methylation level of the other one is also determined, i.e. of 11 and 12; and/or
  • the methylation level of one of Alu CpG dinucleotides 15, 16 or 17 is determined, the methylation level of one or two of the remaining two is also determined, i.e. of 15 and 16, 15 and 17, 16 and 17 or 15 to 17.
  • the methylation levels of the CpG dinucleotides of these groups are preferably summarized, e.g. as a mean, for each group.
  • the methylation profiles of CpG sites within a certain genomic distance are not independent. CpG sites usually have a correlated methylation state, i.e., they are co-methylated.
  • the method of the first aspect also includes co-methylated CpG dinucleotides in particular of LINEl CpG dinucleotide 1 of SEQ ID NO: 1.
  • the CpG dinucleotides within SEQ ID NO: 3 that are co-methylated with LINEl CpG dinucleotide 1 of SEQ ID NO: l are limited to CpG dinucleotides within a range of 1000 nucleotides downstream and upstream of that specific CpG dinucleotide (i.e. posititions 140 to 2141), a range of 500 nucleotides downstream and upstream of that specific CpG dinucleotide (i.e.
  • posititions 640 to 1641 or a range of 100 nucleotides downstream and upstream of that specific CpG dinucleotides (i.e. posititions 1040 to 2241) of SEQ ID NO: 3.
  • "Co-methylated” referring to having the same methylation status (i.e. not methylated or methylated) and with respect to the invention having the same methylation status in the genomic DNA according to first aspect in either subjects having the cancer or not having the cancer.
  • the term "co-hypomethylated” can also be used to define such CpG dinucleotides.
  • co-hypomethylated refers to a methylation level that is substantially the same as the LINEl CpG dinucleotide 1 of SEQ ID NO: 1 methylation level. "Substantially the same” may comprise a variation of +/- 0.1, preferably +/- 0.05 of methylation levels between 0 and 1. Preferably, “substantially the same” refers to the absence of a statistially significant difference. For a description of statistic significance and suitable confidence intervals and p values, see below. Co-methylation can be determined without undue burden by the skilled person, e.g. by NGS sequencing.
  • the cancer preferably is breast cancer, ovarian cancer or pancreatic cancer, most preferably breast cancer.
  • the subject is preferably is selected from the group consisting of laboratory animals (e.g. mouse or rat), domestic animals (including e.g. guinea pig, rabbit, horse, donkey, cow, sheep, goat, pig, chicken, camel, cat, dog, turtle, tortoise, snake, or lizard), or primates including chimpanzees, bonobos, gorillas, and humans. Humans are particularly preferred.
  • laboratory animals e.g. mouse or rat
  • domestic animals including e.g. guinea pig, rabbit, horse, donkey, cow, sheep, goat, pig, chicken, camel, cat, dog, turtle, tortoise, snake, or lizard
  • primates including chimpanzees, bonobos, gorillas, and humans. Humans are particularly preferred.
  • breast cancer or ovarian cancer it is preferred that the subject is female.
  • the subject has an increased risk of having or developing the cancer.
  • the subject has at least one risk factor for having or developing the cancer.
  • the subject has for example at least one risk factor selected from the group consisting of female gender, age of 45 or older (in particular 50 or 55 or older), having a risk-increasing inherited syndrome or predisposition (such as mutation of the BRCA1, BRCA2, ATM, TP53, CHEK2, PTEN, CDH1, STK11 and/or PALB2 gene), family history of breast cancer (in particular first degree relatives), personal history of breast cancer in another part of the breast or in the other breast, ethnicity (in particular White), dense breast tissue (in particular a dense breast on mammogram 1.2 to 2 times of the average breast density of women), benign breast conditions (such as (i) non-proliferative lesions including fibrosis and/or simple cysts, mild hyperplasia, adenosis (non- sclera sing), ductal ectasia, Phyllodes tumor (benign), single papilloma, fat necrosis, periductal fibros
  • the subject has for example at least one risk factor selected from the group consisting of age of 40 or older (in particular of 63 and older and more particular after menopause), female gender, obesity, first full-term pregnancy after age 35 or no pregancy, no use of birth control pills (in particular never or not in the last 3, 4 or 6 months), exposure to fertility drugs (in particular clomiphene citrate), exposure to androgens, estrogen therapy and/or hormone therapy, family history of ovarian cancer, breast cancer, or colorectal cancer (in particular in first degree relatives), having an inherited cancer syndrome or predisposition (such as hereditary breast and ovarian cancer syndrome, PTEN tumor hamartoma syndrome, hereditary nonpolyposis colon cancer, Peutz-Jeghers syndrome, or MUTYH-associated polyposis), personal history of breast cancer, use of talcum powder applied directly to the genital area, high fat diet, use of analgesics, and tobacco consumption (in particular smoking).
  • age of 40 or older in particular of 63 and
  • pancreatic cancer With respect to pancreatic cancer, the subject has for example at least one risk factor selected from the group consisting of male gender, age of 45 or older (in particular 55 or older or 65 or older), asian, hispanic or white origin, tobacco consumption (in particular smoking), heavy alcohol use (as defined above), being overweight or obese, family history of pancreatic cancer (in particular at least two or at least three first degree relatives, e.g.
  • pancreatic cancer an inherited condition selected from the group consisting of Hereditary pancreatitis (HP), Peutz- Jeghers syndrome (PJS), Familial malignant melanoma and pancreatic cancer (FAMM-PC), Hereditary breast and ovarian cancer (HBOC) syndrome, Lynch syndrome, and Li-Fraumeni syndrome (LFS), Familial adenomatous polyposis (FAP); chronic pancreatitis, infection with H. pylori or hepatitis B, and liver cirrhosis.
  • HP Hereditary pancreatitis
  • JS Joint Jeghers syndrome
  • FAMM-PC Familial malignant melanoma and pancreatic cancer
  • HBOC Hereditary breast and ovarian cancer
  • LFS Li-Fraumeni syndrome
  • FAP Familial adenomatous polyposis
  • the sample can be a body fluid sample or a tissue sample.
  • the body fluid sample can be selected from the group consisting of blood, serum, plasma, synovial fluid, urine, saliva, lymphatic fluid, lacrimal fluid, and fluid obtainable from the glands such as e.g. breast.
  • the sample comprises blood cells, so it can be blood (e.g. whole blood) or a blood-derived sample, but is not limited thereto.
  • blood e.g. whole blood
  • peripheral blood e.g. whole peripheral blood
  • the genomic DNA is DNA from blood cells, more preferably peripheral blood cells.
  • Methods for determining the level of cytosine methylation are well-known in the art and the method of the first aspect is not limited to any particular method for determining the level of of cytosine methylation.
  • Examplary methods are COBRA, restriction ligation-mediated PCR, Ms- SNuPE, ion-pair reverse-phase high performance liquid chromatography, denaturing high performance liquid chromatography, any bisulfite sequencing method, e.g. direct bisulfite sequencing with the Sanger method or sequencing methods of the 2 nd or 3 rd generation (NexGen sequencing, NGS), or any pyrosequencing method, DNA sequencing methods that can per se distinguish between methylated and unmethylated cytosines (e.g.
  • Nanopores and/or enzymes used as sensors digital sequencing, mass spectrometry (e.g. MALDI-TOF), QMTM or quantitative real-time PCR, preferably MethyLightTM or HeavyMethylTM, or methylation sensitive restriction enzyme analysis, and any other quantitative methylation determination technique including nanotechnology approaches.
  • mass spectrometry e.g. MALDI-TOF
  • QMTM or quantitative real-time PCR preferably MethyLightTM or HeavyMethylTM, or methylation sensitive restriction enzyme analysis, and any other quantitative methylation determination technique including nanotechnology approaches.
  • detecting cytosine methylation comprises converting, in the genomic DNA, cytosine unmethylated in the 5-position to uracil or another base that does not hybridize to guanine, preferably by bisulfite conversion.
  • the method of the first aspect determines methylation levels rather than simply an absence or presence of methylation. Accordingly, the cytosine methylation level of each of the at least one CpG dinucleotides represents the cytosine methylation of at least a representative large part (depending on the method) or all copies of each of the at least one CpG dinucleotides in the sample.
  • the cytosine methylation level of each of the at least one CpG dinucleotides is the mean cytosine methylation of these copies of each of the at least one CpG dinucleotides in the sample.
  • hypomethylation is a mean methylation that is within the three lower quartiles, preferably the two lower quartiles and more preferably the lowest quartile of the mean methylation of a cancer-positive control.
  • the method of the first aspect can comprise determining the mean cytosine methylation for each of at least two CpG dinucleotides (or at least three, four, etc.), and further comprise determining the overall mean of the mean cytosine methylation of each of the at least two CpG dinucleotides. Examples of this embodiment are shown in Tables 5 and 8.
  • the method of the first aspect comprises the step of comparing the cytosine methylation level to a cancer-negative control methylation level of the same at least one CpG dinucleotide.
  • the cancer-negative control methylation preferably is that of one or more control subjects not having the cancer. Accordingly, the cancer-negative control methylation level is the base-line methylation level and all lower methylation levels are considered as hypomethylation.
  • hypomethylation is a lower mean methylation.
  • a lack of hypomethylation preferably is at least the level of methylation of a subject not having the cancer.
  • the method of the first aspect may comprise the step of comparing the cytosine methylation level to a cancer-positive control methylation level of the same at least one CpG dinucleotide, wherein a lower or equal methylation level is indicative of the presence of the cancer. Also, a higher methylation level can be taken as indicative of the absence of the cancer.
  • the cancer-positive control methylation is preferably that of a plurality of control subjects having the cancer.
  • the step of comparing comprises an age adjustment (i.e. a subject is compared only to controls of the same or a similar age, e.g. +/- 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 years), a cell type adjustment (i.e. only the same cell type of several comprised in the sample is compared), or both.
  • an age adjustment i.e. a subject is compared only to controls of the same or a similar age, e.g. +/- 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 years
  • a cell type adjustment i.e. only the same cell type of several comprised in the sample is compared
  • the method of the first aspect comprises a normalisation of the methylation level, e.g. using the methylation level of one or more CpG dinucleotides that are not differentially methylated between subjects having and not having the cancer.
  • An exemplary gene comprising such CpG sites is actin-B.
  • absolute mean methylation values for the CpG dinucleotides may vary depending on factors such as the detection method or the normalisation used. Therefore, it is difficult to set general absolute methylation level thresholds that are valid for all conditions.
  • a mean methylation of Alu CpG dinucleotide 13 of SEQ ID NO: 2 of 0.65 or lower, preferably 0.62 or lower, as would be determined by MALDI-TOF with age normalisation is indicative of the presence of the cancer.
  • the setting of these thresholds values does not require that the method of the first aspect comprises determining methylation levels by MALDI- TOF and/or an age normalisation, this merely defines thresholds and may require determining concordance values for a different detection method and normalisation that is to be used.
  • the above thresholds for LINE1 CpG dinucleotide 1 of SEQ ID NO: 1 may also be used if the methylation level of a co-methylated CpG dinucleotide (as described herein) is determined.
  • the method of the first aspect may further comprise the detection of additional markers of the cancer.
  • additional markers can be used in addition to one or more of the CpG dinucleotides described above.
  • they are used in addition to LINE1 CpG dinucleotide 1 of SEQ ID NO: 1 (in combination with or without one or more of the remaining INE1 or Alu CpG dinucleotides described).
  • the method of the first aspect further comprises determining the level of cytosine methylation of at least one CpG dinucleotide within and/or gene expression of at least one gene selected from the group consisting of HYAL2, MGRN1, RPTOR, SLC22A18, FUT7, RAPSN, S100P, and DYRK4 in the genomic DNA, wherein hypomethylation and/or increased expression is indicative of the presence of the cancer and the lack hypomethylation and/or decreased or expression is indicative of the absence of the cancer.
  • the method of the first aspect further comprises determining the amount of at least one RNA marker selected from the group consisting of miR-652, spliceosomal RNA Ul 1 or a fragment thereof (previously described as miR-801), miR-376c, miR-376a, miR-127-3p (previously described as miR-127), miR-409-3p (previously described as miR-409) and miR-148b in the sample, wherein an increased amount of the at least one RNA marker is indicative of the presence of the cancer and/or a decreased or normal amount of the at least one RNA marker is indicative of the absence of the cancer.
  • This determination is also described in WO 2016/135168.
  • the method of the first aspect further comprises (next to or without the additional markers described above) determining the amount of at least one miRNA marker selected from the group consisting of miR-328, miR-320, miR-145, miR-339-3p, and miR-193a-3p in the sample, wherein an increased amount of miR-328, miR-145 and/or miR- 339-3p, and/or a decreased amount of miR-320 and/or miR-193a-3p is indicative of the presence of the cancer, and wherein a decreased or normal amount of miR-328, miR-145 and/or miR-339- 3p, and/or an increased or normal amount of miR-320 and/or miR-193a-3p is indicative of the absence of the cancer.
  • Example 2 The utility of the markers is shown in Example 2. Decreased amount/expression or increased amount/expression is to be understood as a comparison to one or more subjects not having the cancer (cancer-negative control). Normal is to be understood as not being statistically significantly different from one or more subjects not having the cancer (cancer- negative control). For a description of statistic significance and suitable confidence intervals and p values, see below.
  • Any additional determination as described above preferably also comprises one or more comparisons (e.g. to cancer-negative or cancer-positive), adjustments and/or normalisation as described above.
  • a comparisons e.g. to cancer-negative or cancer-positive
  • adjustments and/or normalisation as described above.
  • an amount of RNA it is preferred that an RNA that is not differentially expressed in subjects having and not having the cancer is used for normalisation.
  • An example is cel-miR-39.
  • the determination of the amount of RNA described herein usually can use the same sample as the determination of methylation levels, but instead of using cellular nucleic acids, the determination of RNA is directed to cell-free circulating RNA. This means free-floating RNA in the sample rather than RNA isolated from cells. For example, if peripheral blood is the sample, the amount of RNA in the cell-free fraction of the blood, e.g. in serum or plasma, is determined. However, also a different sample can be used to determine the amount of RNA (independent of the sample used for determining the methylation level). Such a different sample is usually a body fluid sample as described above, in particular blood plasma, blood serum, fluid obtainable from the breast glands, or saliva.
  • the amount of a RNA can be determined by techniques well known in the art. Depending on the nature of the sample, the amount may be determined by PCR based techniques for quantifying the amount of a polynucleotide or by other methods like mass spectrometry or (next generation) sequencing.
  • the term "determining the amount of at least one RNA/miRNA", as used herein, preferably relates to determining the amount of each of the RNA separately in order to be able to compare the amount of each RNA to a cancer-positive or -negative control.
  • the present invention relates to a method for diagnosing cancer or for screening for cancer, comprising detecting the cancer according to the first aspect.
  • Diagnosis refers to a determination whether a particular subject does or does not have cancer. A diagnosis by the methods described herein may be supplemented with a further diagnostic means to detect or confirm the presence of the cancer. As will be understood by persons skilled in the art, the diagnosis normally may not be correct for 100% of the subjects, although it preferably is correct. The term, however, requires that a correct diagnosis can be made for a statistically significant part of the subjects.
  • “Screening for cancer” refers to the use of the method of the first aspect with samples of a population of subjects.
  • the subjects Preferably, the subjects have an increased risk for or are suspected of having the cancer.
  • one or more of the risk factors recited herein can be attributed to the subjects of the population.
  • the same one or more risk factors can be attributed to all subjects of the population.
  • the population may be characterized by a certain minimal age (e.g. 50 or older).
  • the term “screening” does not necessarily indicate a definite diagnosis, but is intended to indicate an increased possibility of the presence or of the absence of the cancer. An indicated increased possibility is preferably confirmed using a further diagnostic means.
  • the screening result normally may not be correct for 100% of the subjects, although it preferably is correct. The term, however, requires that a correct screening result can be achieved for a statistically significant part of the subjects.
  • the present invention relates to a method for monitoring a subject having an increased risk of developing cancer, comprising detecting the cancer according to the first aspect repeatedly.
  • one or more of the risk factors recited herein can be attributed to the subject.
  • the method according to the first aspect is carried out periodically over an extended amount of time, e.g. once per year, per two years, per three years, per four years, per five years of per ten years for at least two times, preferably at least three times, at least four times, at least five times or at least ten times. This preferably occurs until the subject no longer has the one or more risk factors, until the cancer is detected (in that case the method of the first aspect can still be carried out for different purposes as described herein, though, e.g. according to the fourth or fifth aspect), until the subject dies or until the monitoring is terminated for any other reason.
  • the present invention relates to a method for monitoring cancer treatment of a subject, comprising detecting the cancer according to the first aspect repeatedly across the treatment period.
  • This relates to the accompaniment of a diagnosed cancer during a treatment procedure or during a certain period of time, typically during at least 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, 5 years, 10 years, or any other period of time.
  • accompaniment means that states of and, in particular, changes of these states of a cancer may be detected, particular based on changes in the amount of methylation/RNA/miRNA in any type of periodical time segment, determined e.g.
  • Amounts or changes in the amounts can also be determined at treatment specific events, e.g. before and/or after every treatment cycle or drug/therapy administration.
  • a cycle is the time between one round of treatment until the start of the next round.
  • Cancer treatment is usually not a single treatment, but a course of treatments. A course usually takes between 3 to 6 months, but can be more or less than that. During a course of treatment, there are usually between 4 to 8 cycles of treatment. Usually a cycle of treatment includes a treatment break to allow the body to recover.
  • the result of the monitoring normally may not be correct for 100% of the subjects, although it preferably is correct.
  • the term requires that a correct result of the monitoring can be achieved for a statistically significant part of the subjects.
  • statistic significance and suitable confidence intervals and p values see below.
  • the present invention relates to a method for assessing the response of a subject to a cancer treatment, comprising detecting the cancer according to the first aspect during and/or after the treatment.
  • Response to treatment refers to the response of a subject suffering from cancer to a therapy for treating said disease.
  • Standard criteria can be used to evaluate the response to therapy include response, stabilization and progression.
  • the response can be a complete response (or complete remission) which is the disappearance of all detectable malignant disease or a partial response which is defined as approximately >50% decrease in the sum of products of the largest perpendicular diameters of one or more lesions (tumor lesions), no new lesions and no progression of any lesion.
  • Subjects achieving complete or partial response are considered “responders", and all other subjects are considered “non- responders”.
  • stabilization is defined as a ⁇ 50% decrease or a ⁇ 25% increase in tumor size.
  • progression is defined as an increase in the size of tumor lesions by > 25% or appearance of new lesions.
  • the present invention relates to a method for treating a subject having cancer detected according to the method of the first aspect, comprising administering a cancer therapy to the subject.
  • a cancer therapy comprising administering a cancer therapy to the subject.
  • Suitable cancer treatments are described below. Definitions given and embodiments described with respect to the first aspect apply also to all other aspects, in as far as they are applicable. Also, definitions and embodiments described below, apply to all methods described above.
  • univariate marker refers to a marker that is determined independently, i.e. not in combination with another marker.
  • multivariate marker refers to a marker that is determined in combination with another marker.
  • “Another marker” with respect to a CpG dinucleotide refers herein to another CpG dinucleotide (preferably of LINEl or Alu). In a broader sense it can also include any other marker, including markers not disclosed herein.
  • level of cytosine methylation refers to the methylation of multiple copies of the same CpG dinucleotide. It is preferably expressed as the mean methylation with a value of 0 to 1, corresponding to 0% to 100% of the multiple copies being methylated.
  • expression level refers to the amount of gene product present in the body or a sample. The expression level can e.g. be measured/quantified/detected by means of the protein or mRNA expressed from the gene. The expression level can for example be quantified by normalizing the amount of gene product of interest present in a sample with the total amount of gene product of the same category (total protein or mRNA) in the same sample or a reference sample (e.g.
  • the expression level can be measured or detected by means of any method as known in the art, e.g. methods for the direct detection and quantification of the gene product of interest (such as mass spectrometry) or methods for the indirect detection and measurement of the gene product of interest that usually work via binding of the gene product of interest with one or more different molecules or detection means (e.g. primer(s), probes, antibodies, protein scaffolds) specific for the gene product of interest.
  • the determination of the level of gene copies comprising also the determination of the absence or presence of one or more fragments (e.g. via nucleic acid probes or primers, e.g. quantitative PCR, Multiplex ligation- dependent probe amplification (MLPA) PCR) is also within the knowledge of the skilled artisan.
  • miRNA refers to at least a mature miRNA, optionally comprising the complete stem-loop. While in Example 2, mature miRNAs are detected, the stem-loop miRNAs can be detected instead.
  • breast cancer is used in the broadest sense and refers to all cancers that start in the breast. It includes the subtypes ductal carcinoma in situ, invasive ductal carcinoma (including tubular carcinoma of the breast, medullary carcinoma of the breast, mucinous carcinoma of the breast, papillary carcinoma of the breast, and cribriform carcinoma of the breast), invasive lobular carcinoma, inflammatory breast cancer, lobular carcinoma in situ, male breast cancer, Paget's disease of the nipple, and Phyllodes tumors of the breast.
  • invasive ductal carcinoma including tubular carcinoma of the breast, medullary carcinoma of the breast, mucinous carcinoma of the breast, papillary carcinoma of the breast, and cribriform carcinoma of the breast
  • invasive lobular carcinoma including tubular carcinoma of the breast, medullary carcinoma of the breast, mucinous carcinoma of the breast, papillary carcinoma of the breast, and cribriform carcinoma of the breast
  • invasive lobular carcinoma including tubular carcinoma of the breast, medu
  • stage 0 (Tis, NO, M0), stage IA (Tl, NO, M0), stage IB (TO or Tl, Nlmi, M0), stage IIA (TO or Tl, Nl (but not Nlmi), M0; or T2, NO, M0), stage IIB (T2, Nl, M0; or T3, NO, M0), stage IIIA (TO to T2, N2, M0; or T3, Nl or N2, M0), stage IIIB (T4, NO to N2, M0), stage IIIC (any T, N3, M0), and stage IV (any T, any N, Ml).
  • ovary cancer or "ovarian cancer” is used in the broadest sense and refers to all cancers that start in the ovaries. It includes the subtypes benign epithelial ovarian tumors, tumors of low malignant potential, and malignant epithelial ovarian tumors.
  • stage IA Tla, NO, MO
  • stage IB Tib, NO, MO
  • stage IC Tic, NO, MO
  • stage IIA T2a, NO, MO
  • stage IIB T2b, NO, MO
  • stage IIIA1 Tl or T2, Nl, MO
  • stage IIIA2 T3a2, NO or Nl, MO
  • stage IIIB T3b, NO or Nl, MO
  • stage IIIC T3c, NO or Nl, MO
  • stage IV any T, any N, Ml.
  • pancreatic cancer is used in the broadest sense and refers to all cancers that start in the pancreas. It includes the subtypes exocrine cancers, endocrine cancers, pancreatoblastoma, sarcomas of the pancreas, and lymphoma. Exocrine cancers include adenocarcinomas, in particular ductal adenocarcinomas, as well as cystic tumours and cancer of the acinar cells. Endocrine cancers include gastrinomas, insulinomas, somatostatinomas, VTPomas, and glucagonomas.
  • stage 0 Tis, NO, MO
  • stage IA Tl, NO, MO
  • stage IB T2, NO, MO
  • stage IIA T3, NO, MO
  • stage IIB Tl-3, Nl, MO
  • stage III T4, any N, MO
  • stage IV any T, any N, Ml
  • TNM classification is a staging system for malignant cancer.
  • TNM classification refers to the 6 th edition of the TNM stage grouping as defined in Sobin et al. (International Union against Cancer (UICC), TNM Classification of Malignant tumors, 6 th ed. New York; Springer, 2002, pp. 191-203).
  • the term "is indicative for” or "indicates” as used herein refers to an act of identifying or specifying the thing to be indicated. As will be understood by persons skilled in the art, such assessment normally may not be correct for 100% of the subjects, although it preferably is correct. The term, however, requires that a correct indication can be made for a statistically significant part of the subjects. Whether a part is statistically significant can be determined easily by the person skilled in the art using several well known statistical evaluation tools, for example, determination of confidence intervals, determination of p values, Student's t-test, Mann- Whitney test, etc. Details are provided in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. The preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%. The p values are preferably 0.05, 0.01, or 0.005.
  • risk with respect to the method for detecting the presence or absence of cancer in a subject refers to the detection of an increased risk of developing the cancer or an increased probability of having it. If the subject already has an increased risk in view of one or more risk factors that can be attributed to it (as defined herein), the 'risk therof refers to a risk that is increased further, i.e. that is in addition to the risk due to those risk factors.
  • treatment or “treating” with respect to cancer as used herein refers to a therapeutic treatment, wherein the goal is to reduce progression of cancer.
  • Beneficial or desired clinical results include, but are not limited to, release of symptoms, reduction of the length of the disease, stabilized pathological state (specifically not deteriorated), slowing down of the disease's progression, improving the pathological state and/or remission (both partial and total), preferably detectable.
  • a successful treatment does not necessarily mean cure, but it can also mean a prolonged survival, compared to the expected survival if the treatment is not applied.
  • the treatment is a first line treatment, i.e. the cancer was not treated previously. Cancer treatment involves a treatment regimen.
  • treatment regimen refers to how the subject is treated in view of the disease and available procedures and medication.
  • cancer treatment regimes are chemotherapy, surgery and/or irradiation or combinations thereof.
  • the early detection of cancer the present invention enables allows in particular for a surgical treatment, especially for a curative resection.
  • treatment regimen refers to administering one or more anti-cancer agents or therapies as defined below.
  • anti-cancer agent or therapy refers to chemical, physical or biological agents or therapies, or surgery, including combinations thereof, with antiproliferative, antioncogenic and/or carcinostatic properties.
  • a chemical anti-cancer agent or therapy may be selected from the group consisting of alkylating agents, antimetabolites, plant alkaloyds and terpenoids and topoisomerase inhibitors.
  • the alykylating agents are platinum-based compounds.
  • the platinum-based compounds are selected from the group consisting of cisplatin, oxaliplatin, eptaplatin, lobaplatin, nedaplatin, carboplatin, iproplatin, tetraplatin, lobaplatin, DCP, PLD-147, JM1 18, JM216, JM335, and satraplatin.
  • a physical anti-cancer agent or therapy may be selected from the group consisting of radiation therapy (e.g. curative radiotherapy, adjuvant radiotherapy, palliative radiotherapy, teleradiotherapy, brachytherapy or metabolic radiotherapy), phototherapy (using, e.g. hematoporphoryn or photofrin II), and hyperthermia.
  • radiation therapy e.g. curative radiotherapy, adjuvant radiotherapy, palliative radiotherapy, teleradiotherapy, brachytherapy or metabolic radiotherapy
  • phototherapy using, e.g. hematoporphoryn or photofrin II
  • hyperthermia e.g. hematoporphoryn or photofrin II
  • Surgery may be a curative resection, palliative surgery, preventive surgery or cytoreductive surgery. Typically, it involves an excision, e.g. intracapsular excision, marginal, extensive excision or radical excision as described in Baron and Valin (Rec. Med. Vet, Special Cane. 1990; 11(166):999-1007).
  • excision e.g. intracapsular excision, marginal, extensive excision or radical excision as described in Baron and Valin (Rec. Med. Vet, Special Cane. 1990; 11(166):999-1007).
  • a biological anti-cancer agent or therapy may be selected from the group consisting of antibodies (e.g. antibodies stimulating an immune response destroying cancer cells such as retuximab or alemtuzubab, antibodies stimulating an immune response by binding to receptors of immune cells an inhibiting signals that prevent the immune cell to attack "own" cells, such as ipilimumab, antibodies interfering with the action of proteins necessary for tumor growth such as bevacizumab, cetuximab or panitumumab, or antibodies conjugated to a drug, preferably a cell- killing substance like a toxin, chemotherapeutic or radioactive molecule, such as Y-ibritumomab tiuxetan, I-tositumomab or ado-trastuzumab emtansine), cytokines (e.g.
  • interferons or interleukins such as INF-alpha and IL-2
  • vaccines e.g. vaccines comprising cancer-associated antigens, such as sipuleucel-T
  • oncolytic viruses e.g. naturally oncolytic viruses such as reovirus, Newcastle disease virus or mumps virus, or viruses genetically engineered viruses such as measles virus, adenovirus, vaccinia virus or herpes virus preferentially targeting cells carrying cancer-associated antigens such as EGFR or HER-2
  • gene therapy agents e.g.
  • DNA or RNA replacing an altered tumor suppressor blocking the expression of an oncogene, improving a subject's immune system, making cancer cells more sensitive to chemotherapy, radiotherapy or other treatments, inducing cellular suicide or conferring an anti- angiogenic effect) and adoptive T cells (e.g. subject-harvested tumor-invading T-cells selected for antitumor activity, or subject-harvested T-cells genetically modified to recognize a cancer-associated antigen) .
  • adoptive T cells e.g. subject-harvested tumor-invading T-cells selected for antitumor activity, or subject-harvested T-cells genetically modified to recognize a cancer-associated antigen
  • the one or more anti-cancer drugs is/are selected from the group consisting of Abiraterone Acetate, ABVD, ABVE, ABVE-PC, AC, AC-T, ADE, Ado- Trastuzumab Emtansine, Afatinib Dimaleate, Aldesleukin, Alemtuzumab, Aminolevulinic Acid, Anastrozole, Aprepitant, Arsenic Trioxide, Asparaginase Erwinia chrysanthemi, Axitinib, Azacitidine, BEACOPP, Belinostat, Bendamustine Hydrochloride, BEP, Bevacizumab, Bexarotene, Bicalutamide, Bleomycin, Bortezomib, Bosutinib, Brentuximab Vedotin, Busulfan, Cabazitaxel, Cabozantinib-S-Malate, CAFCapecitabine, CAPO
  • SEQ IDs referred to in the application The present application refers to SEQ ID NOs 1-7. An overview of these SED ID NOs is given in the following:
  • SEQ ID NO: 1 represents a LINE1 sequence investigated by the inventors.
  • SEQ ID NO: 2 represents an Alu sequence investigated by the inventors.
  • the specific CpG dinucleotides referred to herein have the following positions within SEQ ID NOs 1 and 2: LINE1 (SEQ ID NO: 1): dinucleotide 1 - positions 26/27, dinucleotide 2 - positions 44/45, dinucleotide 3 - positions 49/50, dinucleotide 4 - positions 51/52, dinucleotide 5 - positions 53/54, dinucleotide 6 - positions 60/61, dinucleotide 7 - positions 70/71, dinucleotide 8 - positions 94/95, dinucleotide 9 - positions 120/121, dinucleotide 10 - positions 140/141, dinucleotide 11 - positions 144/145, dinucleotide 12 - positions 158/159, dinucleotide 13 - positions 173/174, dinucleotide 14 - positions 182/183, dinucleotide 15 - positions 194/195,
  • SEQ ID NO: 3 represents the human genomic consensus sequence of LINE1.
  • LINE1 CpG dinucleotide 1 of SEQ ID NO: 1 has the positions 1140/1141 in SEQ ID NO: 3.
  • the other LINE1 CpG dinucleotide have corresponsing positions.
  • SEQ ID NO: 4 represents LINE1 sense primer of Table 3.
  • SEQ ID NO: 5 represents LINE1 antisense primer of Table 3.
  • SEQ ID NO: 6 represents Alu sense primer of Table 3.
  • SEQ ID NO: 7 represents Alu antisense primer of Table 3.
  • SEQ ID Nos 8-12 represent the mature miRNAs miR-328, miR-320, miR-145, miR-339-3p, and miR-193a-3p, respectively.
  • Example 1 Investigation of LINE1 and Alu methylation in peripheral blood from Breast Cancer Patients
  • DNA from whole blood samples was extracted by using QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's recommendations. DNA quality and quantity were measured by the NanoDrop ND- 1000 UV/Vis-Spectralphotometer 3.3 (peqLab, Er Weg, Germany). Aliquots of DNA (500ng) were bisulfite-treated with the EZ- 96 DNA methylation Gold kit (Zymo Research Corporation, Orange, US) according as the description of the manufacturer. Primer design and PCR amplification
  • PCR primers for LINEl and Alu and their amplicon sequences are shown in Tables 3 and 4.
  • PCR reaction was performed in a total volume of 6 ⁇ 1 mix.
  • PCR reaction components included lOng/ ⁇ bisulfite-treated DNA, 10 x CoralLoad Buffer (Qiagen), lOmM dNTPs, ⁇ of each (forward and reverse) primer(Sigma), and 5U HotStar Taq DNA polymerase (Qiagen, Valencia CA).
  • the touch-down PCR profile was 95°C for 5 minutes, denaturation at 94°C for 30 seconds, primer annealing from 59°C to 53°C for 30 seconds, a final extension at 72°C for 1 minute, then 72°C for 5 minutes, 4°C for infinite.
  • 1% agarose gel was used for electrophoresis to inspect PCR products and visualized under ultraviolet light.
  • IQR interquartile range
  • *p values are calculated by multiple logistic regression and adjusted for age and the other CpGs of the table. Significant p values are in bold.
  • *p values are calculated by multiple logistic regression and adjusted for age and the other CpGs of the table. Significant p values are in bold.
  • the inventors also calculated a model with the 7 most important variables plus age (see Table 13).
  • IQR interquartile range
  • OR odds ratio Correlation of LINEl and Am methylation with clinical characteristics of BC patients
  • Her-2 status (181) Her-2 negative (165) 48 0.76 0.92 0.87 0.87 0.71 0.90 0.93 0.88 0.85
  • methylation level of repetitive elements is thought to reflect the average methylation level of genomic DNA
  • the inventors compared the mean methylation of LINEl and Alu in Sequenom data with the mean of all the CpG sites in 450K methylation array.
  • the inventors found statistically significant LINEl hypomethylation in the peripheral blood DNA of BC patients compared with healthy controls, especially for LINEl_CpG_l. They identified inter alia LINEl_CpG_l methylation to be strongly associated with BC. Also for Alu, they observed that single CpG sites were significantly hypomethylated in the peripheral blood DNA from BC cases compared to controls. Furthermore, the results show that the decreased methylation level of inter alia LINEl_CpG_l is associated with an increased BC risk. Also, quartiles of LINEl methylation levels are associated with BC, with an increased risk observed in particular in the lowest quartile compared with those in the highest quartile.
  • RNA extraction including miRNAs
  • a combination of phenol based sample lysis and silica-membrane column extraction was applied.
  • TRIzol LS was added to the liquid samples (volume ratio 3: 1), homogenized by brief vortexing and incubated for 5 minutes at room temperature to permit the nucleoprotein complexes to dissociate.
  • 10 fmol of a synthetic cel-miR-39 oligo was spiked in and 10 ⁇ g of glycogen added.
  • the spiked- in cel-miR-39 was used later on for normalization and glycogen served as an RNA carrier to facilitate the extraction due to expected low RNA yields.
  • Chloroform was added to the lysed samples (volume ratio 1:5) and immediately shaken. Vigorous, simultaneous vortexing of all samples followed before they were incubated at room temperature for 5 minutes. By centrifugation at 12000g for 15 minutes at 8°C the samples separated into three phases. The upper, aqueous phase containing the RNA was transferred to microcentrifugation tubes. The total RNA extraction continued with the components of the miRNeasy Mini kit as per manufacturer's recommendations. The addition of 1.5 volumes of absolute ethanol to the aqueous phase established the conditions for binding of RNA molecules >18 nucleotides in length. To obtain higher RNA concentrations the eluates were re-applied to the same columns and the elution repeated. The eluates were subsequently stored at -80°C until use.
  • TaqMan® Low Density Arrays are 384- well microfluidic cards pre-loaded with dried miRNA- specific TaqMan primers and probes for miRNA quantification.
  • the quantification of miRNAs is based on the two-step RT-PCR described below. After sample preparation, hundreds of miRNAs from one sample are simultaneously reverse transcribed to cDNA in a Megaplex reverse transcription (RT). For samples with low miRNA concentrations, a pre-amplification of cDNA is performed to improve the sensitivity of subsequent miRNA detection. After the cDNA product of one sample is loaded onto the card, the real-time PCR based profiling of miRNAs began.
  • Plasma samples from 10 early stage breast cancer patients and 10 healthy controls were profiled. All patients had a stage I or II invasive ductal carcinoma (IDC), which was ER/PR- positive and HER2-negative. Patients and healthy controls were age-matched. The mean (median) age of the patients was 54 (51) years, while it was 53 (54.5) years for the controls.
  • IDC invasive ductal carcinoma
  • Profiling of plasma samples was carried out utilizing TLDA array Human microRNA Cards A v2.1 and B v2.0 from Applied Biosystems. These arrays measure the expression of 667 human, mature miRNAs from miRBase version v.10.
  • RT Megaplex reverse transcription
  • Pool A and B two pre-amplification reactions
  • TaqMan MicroRNA Arrays Two TaqMan MicroRNA Arrays
  • the RT reaction had a final volume of 7.5 ⁇ and contained a fixed volume of miRNA template (3 ⁇ ) and 4.5 ⁇ of the Megaplex RT reaction mix (see below).
  • the reverse transcription was carried out in a STORM GS2 PCR cycler.
  • RT Megaplex reverse transcription
  • cDNA was pre-amplified in a 25 ⁇ reaction comprising 2.5 ⁇ of the Megaplex RT product and 22.5 ⁇ PreAmp reaction mix described below.
  • the reaction was carried out in a STORM GS2 PCR cycler under thermal-cycling conditions described below.
  • PreAmp pre-amplification reaction mix
  • PreAmp pre-amplification
  • the PreAmp product Prior to loading on the TLDA microfluidic cards, the PreAmp product was diluted with 75 ⁇ of nuclease-free water and stored at -20°C until use (within 2 days of pre-amplification).
  • the TLDA array real-time PCR reaction mix components are listed below. In each of the 8 microfluidic card ports, 100 ⁇ of the TLDA array real-time PCR reaction mix was loaded as described by the manufacturer and the arrays ran in an ABI PRISM 7900HT thermal cycler as specified below.
  • TLDA array real-time PCR reaction mix 450 ⁇ TayMan Universal PCR Master Mix, No AmpErase UNG (2x), 9 ⁇ diluted PreAmp product, 441 ⁇ nuclease free water.
  • Thermal-cycling conditions for the TLDA array real-time PCR reaction 50°C for 2 min, 94.5 °C for 10 min, 40 cycles 97°C for 30 sec and 59.7°C for 1 min, hold at 4°C.
  • Statistical analysis of circulating miRNA profiling data TLDA
  • Raw Ct values from TLDA runs were exported using SDS Relative Quantification Software version 2.2.2 (Applied Biosystems) with automatic baseline and threshold settings.
  • SDS Relative Quantification Software version 2.2.2 Applied Biosystems
  • normalization and quality assessment all 20 samples (10 early stage breast cancer patients and 10 healthy controls) were processed together. The analysis was performed utilizing the statistical computational environment R version 2.11 and the R package HTqPCR version 1.2.0.
  • Ct values larger than 35 were classified as "undetermined" and miRNAs with Ct>35 across all the samples were filtered out from further analysis.
  • PCA principal components analysis
  • TLDA arrays Array-based profiling of circulating miRNAs from plasma (TLDA arrays) and normalization of TLDA data
  • the plasma samples selected for the initial miRNA profiling step intentionally comprised only early stage, i.e. stage I and II, breast cancer patients.
  • the investigated samples were from patients with invasive ductal carcinomas of the luminal subtype (representing the most common type of breast cancer).
  • the patient and control samples selected for the arrays were age-, gender- and ethnicity- matched.
  • two TLDA microfluidic cards were used for each sample measuring the levels of 667 human miRNAs.
  • the array run quality check was conducted by analyzing a heat map of Pearson's correlations of Ct values across samples and principal components analysis (PCA) plots for dates of miRNA extraction, pre- amplification and TLDA runs.
  • PCA principal components analysis
  • the Ct values formed two peaks before and after normalization.
  • On microfluidic card A most of the miRNAs displayed Ct values around 30.
  • the other peak around Ct value of 40 represents the fraction of undetected miRNAs.
  • On card B most miRNAs were present at lower levels compared to the miRNAs from card A, which resulted in a shift of the first peak to approximately a Ct value of 32.

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Abstract

La présente invention concerne le domaine de la pharmacogénomique et en particulier la détection de la présence de méthylation CpG et de miARN dans le sang pour la détection du cancer. Cette détection est utile pour un diagnostic minimalement invasif du cancer ainsi que pour surveiller le traitement du cancer et évaluer une réponse au traitement. L'invention concerne également des méthodes appropriées à cette fin.
PCT/EP2018/079035 2017-10-23 2018-10-23 Nouveaux marqueurs dérivés du sang pour la détection du cancer WO2019081507A1 (fr)

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CN113186279A (zh) * 2020-01-14 2021-07-30 南京腾辰生物科技有限公司 用于辅助诊断癌症的透明质酸酶甲基化标志物及试剂盒
CN113215252A (zh) * 2020-02-05 2021-08-06 南京腾辰生物科技有限公司 用于辅助诊断癌症的甲基化标志物
CN113215252B (zh) * 2020-02-05 2024-04-30 腾辰生物科技(上海)有限公司 用于辅助诊断癌症的甲基化标志物
CN113528636A (zh) * 2020-04-16 2021-10-22 南京腾辰生物科技有限公司 钙结合蛋白基因甲基化作为心脑血管疾病早期诊断的潜在标志物
CN113528636B (zh) * 2020-04-16 2024-04-30 腾辰生物科技(上海)有限公司 钙结合蛋白基因甲基化作为心脑血管疾病早期诊断的潜在标志物
CN114480630A (zh) * 2020-10-26 2022-05-13 南京腾辰生物科技有限公司 用于辅助诊断癌症的一种甲基化标志物

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