[go: up one dir, main page]

WO2012031329A1 - Analyse destinée à la détection et à la surveillance du cancer - Google Patents

Analyse destinée à la détection et à la surveillance du cancer Download PDF

Info

Publication number
WO2012031329A1
WO2012031329A1 PCT/AU2011/001161 AU2011001161W WO2012031329A1 WO 2012031329 A1 WO2012031329 A1 WO 2012031329A1 AU 2011001161 W AU2011001161 W AU 2011001161W WO 2012031329 A1 WO2012031329 A1 WO 2012031329A1
Authority
WO
WIPO (PCT)
Prior art keywords
methylation
loci
leukemia
geneid
dna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/AU2011/001161
Other languages
English (en)
Inventor
Nicholas Chau Lun Wong
Jeffrey Mark Craig
Richard Eric Saffery
David Ashley
Justin Bedo
Adam Kowalczyk
Qiao WANG
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Murdoch Childrens Research Institute
Data61
Original Assignee
Murdoch Childrens Research Institute
National ICT Australia Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from AU2010904085A external-priority patent/AU2010904085A0/en
Application filed by Murdoch Childrens Research Institute, National ICT Australia Ltd filed Critical Murdoch Childrens Research Institute
Publication of WO2012031329A1 publication Critical patent/WO2012031329A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • C12Q2600/118—Prognosis of disease development
    • 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
    • C12Q2600/136—Screening for pharmacological compounds
    • 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
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the present disclosure relates generally to an assay for the determination of epigenetic profiles including epigenetic profiles associated with a pathological condition.
  • the present disclosure teaches an assay to detect epigenetic profiles associated with cancer including its form or type, state and minimum residual disease status.
  • the assay enabled herein further identifies and monitors forms and sub-types of leukemia and other hematological malignancies. Kits and assays for medicaments are also taught herein. Methods for screening agents which modulate methylation are also enabled.
  • Cancer is one of the leading causes of mortality and morbidity across all societies and ethnic groups. Cancer has a complex etiology influenced genetic and environmental pressures. In fact, cancer can be considered a broad spectrum of pathological conditions. [0006] One type of cancer is the hematological form, leukemia. Leukemia is classified on the cell type which becomes malignant.
  • ALL acute lymphoblastic leukemia
  • AML acute myeloid leukemia
  • CML chronic myeloid leukemia
  • CLL chronic lymphocytic leukemia
  • ALL is the most prevalent of the childhood malignancies in developed countries. Although current treatment protocols give rise to a proportion of 5 -year event free survival outcome, many patients still succumb to the disease, relapse or experience secondary effects of the treatment due to their non-specific nature. It is unclear how leukemia arises and the mechanisms involved in this hematological malignancy. It is also difficult if not impossible to select treatment protocols based on a prediction as to their likely efficacy.
  • MRD minimum residual disease
  • DNA methylation plays a role in the regulation of gene expression in higher organisms.
  • the importance of DNA methylation has been highlighted by its involvement in several human diseases.
  • Methylation of cytosine at the 5' position is a widely known covalent modification of human genomic DNA.
  • methylation of CpG islands within regulatory regions of the genome appears to be highly tissue specific.
  • methylation of cytosines outside CpG islands is also important. These regions, within 2kb of CpG islands, have been named “shores” or “island shores” (Irizarry et al., Nature Genetics 47(2,1: 178-186, 2009).
  • Methylation modifications which are also potentially important include the generation of hydroxymethylcytosines and other base methylations as well as RNA methylation.
  • SEQ ID NO Nucleotide and amino acid sequences are referred to by a sequence identifier number (SEQ ID NO).
  • the SEQ ID NOs correspond numerically to the sequence identifiers ⁇ 400>1 (SEQ ID .NO:l), ⁇ 400>2 (SEQ ID NO:2), etc.
  • a summary of the sequence identifiers is provided in Table 1.
  • a sequence listing is provided after the claims.
  • loci includes genes associated with (i) cell fate commitment; (ii) transcription factor activity; (iii) DNA binding; (iv) subcellular location; and (v) transcription regulator activity. Table 2 lists examples of genes in each of these classes.
  • cancer includes hematological malignancies such as leukemias.
  • leukemias include all forms, types and sub-types of leukemias including leukemias which have a high frequency of onset during childhood years (pediatric leukemias) or later onset leukemias (adult leukemias).
  • the present disclosure is instructional of an assay to detect an epigenetic profile indicative of a form or type (including sub-type), state or MRD status of a cancer including leukemia.
  • Such leukemias include acute lymphatic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), small lymphocytic lymphoma (SLL), chronic lymphocytic leukemia (CLL), acute monocytic leukemia (AMOL), Hodgkin's lymphomas (all types), non-Hodgkin's lymphomas (all types) and other lymphoid and myeloid malignancies.
  • ALL acute lymphatic leukemia
  • AML acute myeloid leukemia
  • CML chronic myeloid leukemia
  • SLL small lymphocytic lymphoma
  • CLL chronic lymphocytic leukemia
  • AMOL acute monocytic leukemia
  • the epigenetic profile is also informative as to the spectrum of cancer disease conditions including its various forms, types, sub-types and is useful in monitoring treatment protocols or patients after treatment. Identification of the minimum residual disease (MRD) is important in the decision process in relation to undertaking more or less intensive or toxic therapy and hence is useful for prognosis and tailored therapy on a case by case basis.
  • MRD minimum residual disease
  • An example of epigenetic change is the extent of change in methylation or change in distinction of methylation sites in one or more regions in one or more of the genetic loci associated with (i) cell fate commitment; (ii) transcription factor activity; (iii) DNA binding; (iv) subcellular location; and (v) transcription regulator activity. Examples are listed in Tables 2 and 3.
  • “one or more” includes from 1 to a number which provides 100% confidence that the change in methylation or other epigenetic marker is associated with a disease condition. In an embodiment this range is from 1 to 16 including 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 different genetic loci or 1 to 16 different regions in any one or more of the listed genetic loci. Greater than 16 loci or regions can nevertheless be measured such as from 16 to 1000.
  • a method for identifying an epigenetic profile in the genome of a cell indicative of a cancerous condition or a predisposition thereto comprising screening for the extent of change in epigenetic profile relative to a control or normal/non-cancer tissue within or proximal to a locus selected from one or more loci associated with (i) cell fate commitment; (ii) transcription factor activity; (iii) DNA binding; (iv) subcellular location; and (v) transcription regulator activity wherein a change in the epigenetic profile in or proximal to one or more loci is indicative of the presence of a cancerous condition or a propensity to develop same.
  • Another aspect taught herein is a method for identifying a methylation profile in the genome of a cell indicative of a cancerous condition or a predisposition thereto, the method comprising screening for a change relative to a control or normal/non-cancer tissue in the extent of methylation within or proximal to a locus selected from one or more of the loci associated with (i) cell fate commitment; (ii) transcription factor activity; (iii) DNA binding; (iv) subcellular location; and (v) transcription regulator activity wherein a change in the extent of methylation in or proximal to one or more loci is indicative of the presence of a cancerous condition or a propensity to develop same.
  • the present specification is further instructional of a method for identifying an epigenetic profile in the genome of a cell indicative of a cancerous condition or a predisposition thereto, the method comprising screening for a change relative to a control or normal/non-cancer tissue in the extent of change in epigenetic profile within or proximal to from 1 to 16 regions in one or more of the loci associated with (i) cell fate commitment;
  • transcription factor activity (ii) transcription factor activity; (iii) DNA binding; (iv) subcellular location; and (v) transcription regulator activity wherein a change in the epigenetic profile in or proximal to one or more regions is indicative of the presence of a cancerous condition or a propensity to develop same.
  • a still further aspect enabled herein is a method for identifying a methylation profile in the genome of a cell indicative of a cancerous condition or a predisposition thereto, the method comprising screening for a change relative to a control or normal/non- cancer tissue in the extent of methylation within or proximal to from 1 to 16 regions in one or more of the loci associated with (i) cell fate commitment; (ii) transcription factor activity; (iii) DNA binding; (iv) subcellular location; and (v) transcription regulator activity wherein a change in the extent of methylation in or proximal to one or more regions is indicative of the presence of a cancerous condition or a propensity to develop same.
  • genes or loci in each class of associated with are provided in Table 2.
  • Particular loci which are differentially subject to epigenetic change are listed in Table 3.
  • proximal is meant a region up to approximately lOOOkb up- or down-stream from the 5' or 3' end of a locus and includes regulatory elements within this region.
  • epigenetic change includes a change in epigenetic profile.
  • epigenetic profile includes epigenetic modifications such as methylation including hypermethylation and hypomethylation, RNA/DNA interactions, expression profiles of non-coding RNA, histone modification, changes in acetylation, obiquitylation, phosphorylation and sumoylation, as well as chromatin altered transcription factor levels and the like leading to activation or deactivation of genetic locus expression.
  • the extent of methylation, RNA/DNA interaction and non-coding RNA expression are determined as well as any changes therein.
  • the epigenetic modification is an increase or decrease in methylation or an alteration in distribution of methylation sites or other epigenetic sites.
  • Methods includes methylation of any base in DNA or RNA including methylation of cytosine, hydroxymethylation of cytosine, 5-methylcytosine and methylation of adenine.
  • the present disclosure teaches a method for detecting methylation in DNA or RNA associated with a spectrum of cancerous conditions including pediatric and adult leukemia or their various types and sub-types.
  • Reference to a "cancerous condition” includes hematological malignancies and tissue cancers, such as leukemias, sarcomas, carcinomas and other tumors.
  • a “leukemia” may, therefore, be of any hematological type or sub-type and extends to pediatric and adult leukemias.
  • the epigenetic profile is determined in the genome of a cell or sub-population of cells of a subject. Any cell may be tested including but not limited to blood cells, cerebrospinal fluid (CSF) cells, bone marrow cells, buccal cells and cells from rectal swabs or feces. Cells from pre-natal tissues and embryos may also be tested. In addition, cell free DNA or RNA circulating in whole blood, serum or plasma may also be tested.
  • CSF cerebrospinal fluid
  • Circulating DNA or RNA may also be tested in other fluids such as urine, pus, respiratory fluid, lymph fluid, feces, bile, saliva, sputum, semen, vaginal flow, cerebral spinal fluid, brain fluid, ascites, milk, secretions from the genitourinary tract and a lavage of a tissue or organ (e.g. a lung).
  • fluids such as urine, pus, respiratory fluid, lymph fluid, feces, bile, saliva, sputum, semen, vaginal flow, cerebral spinal fluid, brain fluid, ascites, milk, secretions from the genitourinary tract and a lavage of a tissue or organ (e.g. a lung).
  • DNA or RNA methylation is detected by chemical conversion or methylation specific or sensitive restriction enzymes.
  • Uracil has the sariie base paring behaviour as thymine. It therefore forms a base pair with adenine.
  • 5-Methylcytosine on the other hand, base pairs with guanine. Methylated and unmethylated cytosines can therefore be differentiated. Either approach typically employs a PCR step.
  • the present disclosure further enables a method for monitoring the treatment of a cancerous condition in which the treatment modulates the epigenetic profile of one or more loci listed in Table 2, the method comprising monitoring for a change relative to a control or a pre- and post-treatment sample in the epigenetic profile within or near the locus or loci.
  • the extent of change in methylation profile is determined.
  • the sensitivity of the subject assay allows for determination of MRD at a frequency down to about 10 "4 to about 10 "8 (i.e. one cell per 10 ⁇ to about 10 '8 cells) which enables better prognostic determinations.
  • identification of malignant cells in treated patients or patients in remission allows for the selection of more intensive or less toxic therapies.
  • the present disclosure also teaches for the use of an epigenetic profile in one or more loci listed in Table 2 in the manufacture of an assay to identify an epigenetic profile of a cancerous pathological condition.
  • the epigenetic profile includes the determination of extent of epigenetic change such as extent of methylation in any base in DNA or RNA including in cytosine bases.
  • the cytosines include those in CpG and CpNpG islands and shores and in non-CpG and CpNpG islands and shores.
  • locus or loci includes coding and non-coding regions (e.g. promoter regions, 5' non-coding regions, exons, introns and 3' non-coding regions).
  • the region encompassed by a locus includes its coding sequence, promoter and up- or down-stream regulatory elements typically within approximately lOOOkb of a transcriptional start site or transcriptional termination signal. Hence, this lOOOkb region is regarded as the region proximal to the locus.
  • the assay enabled herein may also be used alone or in combination with assays to detect gene expression transcription of a locus associated with (i) cell fate commitment; (ii) transcription factor activity; (iii) DNA binding; (iv) subcellular location; and (v) transcription regulator activity.
  • loci are provided in Tables 2 and 3.
  • the assay taught herein is also useful in epidemiological studies of different ethnic populations with cancer.
  • the present disclosure further provides a method of identifying an epigenetic profile in populations of subjects indicative of a cancerous pathological condition, the method comprising screening for a change relative to a control in a statistically significant number of subjects the extent of epigenetic profile in one or more loci listed in Table 2 or 3 wherein a significant difference in the extent of epigenetic change compared to a control is indicative of the presence of the pathological condition or a propensity to develop same.
  • the assay may comprise the further step of determining the extent of expression such as by quantitative reverse transcriptase PCR (qRTPCR), TaqMan, gene expression micro arrays or by Northern or Western blot analysis.
  • the epigenetic change is extent of methylation or distribution of methylation sites.
  • the present disclosure further enables a method for screening for an agent which modulates to epigenetic profile of one or more loci listed in Table 2 or 3 in the presence or absence of an agent to be tested, wherein an agent is selected if it induces a change in the epigenetic profile.
  • An agent which modulates methylation includes those which inhibit the DNA methyltransferase enzymes (DNMT1, DNMT2, DNMT3a, DNMT3b).
  • a further embodiment taught herein is a kit for use in the above methods comprising primers to amplify a region within a locus or loci listed in Table 2 or 3 for detection of epigenetic change such as methylation profile in DNA.
  • the kit may also be adopted for use in a multiplex assay.
  • the kit contains components to amplify a genomic region and conduct a methylation assay.
  • ACADL BGLAP, CDH22, CELSR1, CPXM2, CXCL1, CYR61, DHCR24, DMRT3, EGR4, ELOVL4, EPOR, FOXE3, GALR1, GUCY1A2, HS3ST2, KCN 5, KISS1, L3MBTL4, LA A1, LGI2, LPL, LY86, MAG, MMP11, MPST, MS4A7, MSX2, MY03A, MYOD1, NELL1, NINJ1, NKX2-8, NPTX2, OGDHL, ONECUT2, PDE10A, P DREJ, PLD4, PNMA2, PPARG, PRLHR, PRSS12, PTFIA, RBP1, RIMS4, SALL3, SCARF1, SCRN1, SFRP1, SH3GL2, SIXl, SLC18A3, SLC22A3, SLC5A7, SLC8A2, SNAP91, SOX1, SOX17, SOX9, SSTR4, TCL1A, TFAP
  • Table 2 lists genetic loci classed by function.
  • the class of function is (i) cell fate commitment; (ii) transcription factor activity; (iii) DNA binding; (iv) subcellular location; and (v) transcription regulator activity differentially methylated between leukemia and controls.
  • Table 3 lists all the genes identified herein as being subject to differentia] epigenetic regulation. Loci are selected using algorithmic models to separate leukemia bone marrow from normal bone marrow, of which any combination of 16 genes can determine this 100% of the time. Algorithimic models include the Linear Models for Microarray Analysis (LIMMA) model, the Recursive Feature Elimination- Support Vector Machine (RFE-SVM) model and Centroid model. Top candidate loci are those which overlapped all three models. These models could also be used to separate leukemia cases with poor outcome from those with a good outcome. [0033] A list of abbreviations used herein is provided in Table 4.
  • a OL Acute monocytic leukemia
  • C-phosphate-G CpG Cytosine and guanine separated by phosphate
  • N any nucleotide but guanine.
  • the cytosine and N nucleotide are phosphorylated.
  • Figure 1 is a graphical representation showing correlation between methylation values of CpG units from bone marrow samples flash frozen or subject to smearing, staining with Giemsa and mounting with DPX on a microscopic slide.
  • Figure 2 is a schematic representation of hierarchical clustering of genes associated with leukemia and non-leukemia cases. This heatmap is provided in black and white. Color prints of the heatmaps are available from the patentee upon request (black represents methylated DNA 50-100% while white represents 0-50% and gradients thereof).
  • Figure 3 is a graphical representation showing correlation between methylation of FOXE3 and model cancer cell lines.
  • Figure 4 is a graphical representation showing correlation between methylation of TLX3 and cancer cell lines.
  • FIG. 5 is a diagrammatic representation showing the single allele base extension reaction (SABER):-
  • A Primers are initially designed to bisulphite converted DNA (not shown), amplifying a region of interest (approximately 100-300bp).
  • B An extension primer is then designed to hybridize within the amplified region. The extension primer is designed up to the CpG site of interest.
  • the SABER approach differs from normal base extension (C) as only one nucleotide is added in the extension reaction. The primer then extends over the CpG site and terminates following the incorporation of a single nucleotide.
  • the extended primers are then assayed by Mass spectrometry (D).
  • Figure 6 is a representation of an unsupervised clustering heatmap plot of the DNA methylation beta-values from the Illumina Infinium HumanMethylation27 BeadArray of 1 15 ALL-specific probes identified using three supervised learning methods (x-axis). Three distinct clusters comprising the leukemia, remission/non-leukemic and the cell lines are apparent (y-axis). The majority of these probes are hypermethylated in all leukemic samples analyzed. These clusters remained when all 14,876 probes, retained after p- detection cutoff, were taken into account ( Figure 9).
  • Figure 7 is a representation of heatmap plot of SEQUENOM EpiTYPER DNA methylation results generated from 85 cases of B-Cell ALL with matching leukemic and remission bone marrow samples, controls (DONOR) and cancer cell line (REH). DNA methylation data for a total of 103 CpG sites encompassing 16 probes we selected by Infinium analysis are shown here (x-axis). DNA hypermethylation of 77/85 (91%) leukemic bone marrow samples was observed regardless of ALL subtype confirming the existence of a DNA methylation signature associated with leukemia.
  • Figure 8 is a representation showing ALL is associated with an increase in average promoter methylation. Average global promoter DNA methylation levels of bone marrow according to sample group. The overall average beta-value of probes that passed stringent quality control for all samples within a sample group are displayed. Bars represent standard deviation.
  • Figure 9 shows unsupervised clustering of Infinium beta- values of 14,876 probes accurately delineates disease free tissue from leukemia. Heatmap plot of unsupervised hierarchical clustering of the beta-values of 14,876 probes passing stringent quality control.
  • FIG 10 is a representation showing the performance and the average area under the receiver operating characteristics (ROC) curve (AROC) curves of the supervised learning methods applied to the data for DNA methylation profiling, (a) Centroid, (b) RFE- SVM and (c) LIMMA plots are depicted.
  • the average accuracy of classification (accuracy) and the average area under the receiver operating characteristics (ROC) curve (AROC) are plotted against the number of features included in the classification.
  • the error bars represent estimated 95% confidence intervals for the obtained results.
  • Accuracy and AROC reached 100% after 16 features for the Centroid method. Whilst the same accuracy and AROC level was achieved in less than 4 features using RFE-SVM and LIMMA.
  • Figure 11 is a representation of unsupervised hierarchical clustering heatmap of SEQUENOM EpiTYPER results. 163 paediatric leukemia cases were analyzed at three loci Corf76, FBX039 and MYOD1 for DNA methylation. Matching leukaemic and remission bone marrow samples were analyzed from each case. Leukemia subtype for each diagnosed case is also depicted and illustrates a hypermethylation signature associated with leukaemic bone marrow.
  • Figure 12 is a graphical representation showing results from the modified multiplex methylation MALDI-TOF technique. Methylation is a measure of the of amount of extension primer that has been extended (extension occuring only on the methylated template) over the total extension primer input into each reaction. TABLE 5
  • NCCIT Germ Cell Tumor
  • the present disclosure teaches a method for identifying an epigenetic profile associated with indicative, instructive or informative of a pathological condition associated with cancer.
  • the epigenetic profile is in loci associated with one or more of (i) cell fate commitment; (ii) transcription factor activity; (iii) DNA binding; (iv) subcellular location; and (v) transcription regulator activity. Particular examples are listed by function in Table 2.
  • the pathological condition may also be described as a cancerous condition or cancer such as a hematological or blood born cancer (e.g. leukemia) or solid cancerous tumors or various types and sub-types thereof.
  • leukemias, sarcomas, carcinomas and the like including hematological malignancies of the blood, bone marrow and lymph nodes are encompassed by a “cancer” or “cancerous condition” as are there various types and subtypes.
  • epigenetic change includes a change in epigenetic profile.
  • epigenetic profile includes epigenetic modifications such as methylation including hypermethylation, hypomethylation and hydroxymethylation, RNA/DNA interactions, expression profiles of non-coding RNA, histone modification, changes in acetylation, obiquitylation, phosphorylation and sumoylation, as well as chromatin altered transcription factor levels and the like leading to activation or deactivation of genetic locus expression.
  • the extent of methylation, RNA/DNA interaction and non-coding RNA expression are determined as well as any changes therein.
  • the epigenetic modification is an elevation in methylation, an increase or decrease in methylation or an alteration in distribution of methylation sites.
  • types of leukemia include acute lymphatic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML) and chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), acute monocytic leukemia (AMOL), Hodgkin's lymphomas (all types) and non- Hodgkin's lymphomas (all types).
  • Sub-types include forms of leukemia having an age- related onset bias such as pediatric leukemias and adult leukemias.
  • Hematological malignancies include malignancies of either myeloid or lymphoid lineages including lymphomas, lymphocytic leukemias and myelomas (lymphoid lineage malignancies) and acute and chronic myelogenous leukemia, myelodysplasia syndromes and myeloproliferative diseases (myeloid lineage malignancies).
  • the epigenetic profile is in one or more regions in one or more loci listed in Table 2 or 3. These loci are grouped into five classes based on function: i.e. (i) cell fate commitment; (ii) transcription factor activity; (iii) DNA binding; (iv) subcellular location; and (v) transcription regulator activity.
  • Reference to a "locus” or “loci” includes promoter, intron, exon and non-encoding 3' and 5' regions proximal to the locus which regions are up to approximately lOOOkb upstream and downstream of the locus as well as known and yet- to-be-defined regulatory elements associated with the gene.
  • the lOOOkb region is referred to herein as being "proximal" to a locus.
  • the epigenetic profile enables determination of minimal residual disease (MRD) which comprises malignant cells at a frequency down to 10 "3 to about 10 "8 including frequencies of 10 "5 , 10 "6 and 10 "7 and frequencies inbetween (i.e. one cancer cell per 10 3 to 10 8 cells).
  • MRD minimal residual disease
  • the epigenetic profile is determined in the genome of a cell or sub-population of cells of a subject. Any cell may be tested including but not limited to blood cells, cerebrospinal fluid (CSF) cells, bone marrow cells, buccal cells and cells from rectal swabs or feces. Cells from pre-natal tissues and embryos may also be tested. In addition, cell free DNA or RNA circulating in whole blood, serum or plasma may also be tested.
  • CSF cerebrospinal fluid
  • Circulating DNA or RNA may also be tested in other fluids such as urine, pus, respiratory fluid, lymph fluid, feces, bile, saliva, sputum, semen, vaginal flow, cerebral spinal fluid, brain fluid, ascites, milk, secretions from the genitourinary tract and a lavage of a tissue or organ (e.g. a lung).
  • fluids such as urine, pus, respiratory fluid, lymph fluid, feces, bile, saliva, sputum, semen, vaginal flow, cerebral spinal fluid, brain fluid, ascites, milk, secretions from the genitourinary tract and a lavage of a tissue or organ (e.g. a lung).
  • the epigenetic profile is extent of methylation or distribution in methylation sites including an increase or decrease in this extent.
  • Methylation includes methylation of any base in DNA or RNA including methylation of cytosine, hydroxymethylation of cytosine and methylation of adenine.
  • a method for identifying an epigenetic profile in the genome of a cell indicative of a cancerous condition or a predisposition thereto comprising screening for the extent of change in epigenetic profile relative to a control or normal/non-cancer tissue within or proximal to a locus selected from one or more of the loci associated with (i) cell fate commitment; (ii) transcription factor activity; (iii) DNA binding; (iv) subcellular location; and (v) transcription regulator activity wherein a change in epigenetic profile in or proximal to one or more loci is indicative of the presence of a cancerous condition or a propensity to develop same.
  • Another aspect taught herein is a method for identifying a methylation profile in the genome of a cell indicative of a cancerous condition or a predisposition thereto, the method comprising screening for a change relative to a control or normal/non-cancer tissue in the extent of methylation within or proximal to a locus selected from one or more of the loci associated with (i) cell fate commitment; (ii) transcription factor activity; (iii) DNA binding; (iv) subcellular location; and (v) transcription regulator activity wherein a change in the extent of methylation in or proximal to one or more loci is indicative of the presence of a cancerous condition or a propensity to develop same.
  • the present specification is further instructional of a method for identifying an epigenetic profile in the genome of a cell indicative of a cancerous condition or a predisposition thereto, the method comprising screening for a change relative to a control or normal/non-cancer tissue in the extent of change in epigenetic profile within or proximal to a locus selected from 1 to 16 regions in one or more of the loci associated with (i) cell fate commitment; (ii) transcription factor activity; (iii) DNA binding; (iv) subcellular location; and (v) transcription regulator activity wherein a change in the epigenetic profile in or proximal to one or more regions is indicative of the presence of a cancerous condition or a propensity to develop same.
  • a still further aspect enabled herein is a method for identifying a methylation profile in the genome of a cell indicative of a cancerous condition or a predisposition thereto, the method comprising screening for a change relative to a control or normal/non- cancer tissue in the extent of methylation within or proximal to from 1 to 16 regions in one or more of the loci associated with (i) cell fate commitment; (ii) transcription factor activity; (iii) DNA binding; (iv) subcellular location; and (v) transcription regulator activity wherein a change in the extent of methylation in or proximal to one or more loci is indicative of the presence of a cancerous condition or a propensity to develop same.
  • Examples of loci grouped by function are listed in Table 2. All loci identified herein are listed in Table 3.
  • a method for identifying an epigenetic profile in the genome of a cell indicative of a cancerous condition or a predisposition thereto comprising screening for the extent of change in epigenetic profile relative to a control or normal/non-cancer tissue within or proximal to a locus selected from one or more of the loci listed in Table 2 or 3 wherein a change in epigenetic profile in or proximal to one or more loci is indicative of the presence of a cancerous condition or a propensity to develop same.
  • Another aspect taught herein is a method for identifying a methylation profile in the genome of a cell indicative of a cancerous condition or a predisposition thereto, the method comprising screening for a change relative to a control or normal/non-cancer tissue in the extent of methylation within or proximal to a locus selected from one or more of the loci listed in Table 2 or 3 wherein a change in the extent of methylation in or proximal to one or more loci is indicative of the presence of a cancerous condition or a propensity to develop same.
  • the present specification is further instructional of a method for identifying an epigenetic profile in the genome of a cell indicative of a cancerous condition or a predisposition thereto, the method comprising screening for a change relative to a control or normal/non-cancer tissue in the extent of change in epigenetic profile within or proximal to a locus selected from 1 to 16 regions in one or more of the loci listed in Table 2 or 3 wherein a change in the epigenetic profile in or proximal to one or more regions is indicative of the presence of a cancerous condition or a propensity to develop same.
  • a still further aspect enabled herein is a method for identifying a methylation profile in the genome of a cell indicative of a cancerous condition or a predisposition thereto, the method comprising screening for a change relative to a control or normal/non- cancer tissue in the extent of methylation within or proximal to from 1 to 16 regions in one or more of the loci listed in Table 2 or 3 wherein a change in the extent of methylation in or proximal to one or more loci is indicative of the presence of a cancerous condition or a propensity to develop same.
  • the present disclosure also teaches the use of an epigenetic profile within or proximal to a locus selected from one or more of the list in Table 2. or 3 in the manufacture of an epigenetic assay to provide data which are indicative of the presence of a cancerous condition or a propensity to develop a cancerous condition.
  • the epigenetic assay determines extent of methylation and or distribution of methylation sites within or proximal to the locus. In an embodiment, from 1 to the number of loci or regions within a locus required to achieve a 100% confidence level that a subject has or does not have cancer are selected. In an embodiment, this number is 16.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15 or 16 different loci, or 1 to 16 different regions within a single locus, or a combination of both are assayed for any epigenetic change such as change in methylation profile.
  • the loci are associated with (i) cell fate commitment; (ii) transcription factor activity; (iii) DNA binding; (iv) subcellular location; and (v) transcription regulator activity.
  • the epigenetic profile enables the determination of MRD with high sensitivity (down to 10 "3 to 10 "8 ) in a patient.
  • the identification of MRD facilitates the decision process on whether more intensive therapy is required, such as more toxic therapy, a bone marrow transplant or stem cell therapy or whether an alternative type of therapy is required. Hence, the MRD influences the prognosis of a cancer in a subject.
  • a method for determining MRD in a subject comprising determining an epigenetic profile in or proximal to one or more loci listed in Table 2 or 3 wherein an elevation or a decrease in epigenetic change of a specific gene or genes, relative to disease free control tissue is an indication of the MRD.
  • the present disclosure further enables a method for determining MRD in a subject, the method comprising determining a change in methylation profile in or proximal to one or more loci or in one or more regions within the one or more loci listed in Table 2 or 3 wherein an elevation or a decrease in extent of methylation is an indication of the MRD.
  • methylation profile means extent of and or distribution of methylation sites within multiple loci, a single locus or a combination of both.
  • a method for determining MRD in a subject comprising determining an epigenetic profile in or proximal to from 1 to 16 loci listed in Table 2 or in 1 to 16 regions within one or more loci listed in Table 2 or 3 wherein an elevation or a decrease in extent of epigenetic change is an indication of the MRD.
  • Yet a further aspect enabled herein is a method for determining MRD in a subject, the method comprising determining a methylation profile in or proximal from 1 to 16 loci listed in Table 2 or in 1 to 16 regions within one or more loci listed in Table 2 or 3 wherein an elevation or a decrease in extent of methylation is an indication of the MRD.
  • 16 different loci or 16 different regions within one or more loci provides a 100% confidence level of the presence or absence of cancer in a subject.
  • the present disclosure contemplates confidence controls of from about 40% to 100% including 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100%.
  • loci or regions within one or more loci may be selected to provide from about 40% to about 100% confidence level of the presence or absence of cancer. More than 16 loci or regions may be assayed such as from 16 to 1000 or greater. This may occur when an array is used such as in solid phase amplification using from 1 to 1000 immobilized primers on, for example, a chip.
  • Reference to “1 to 1000” include “16 to 1000” means 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 110, 111, 112, 113, 114, 115,
  • a method wherein the extent of methylation provides a quantitative or semi-quantitative or qualitative indication of malignant cells at a frequency down to 10 "3 to 10 "8 , including 10 "5 , 10 "6 and 10 '7 and frequencies inbetween.
  • the method described herein may also be used in conjunction with other assays such as quantitative Reverse transcriptase PCR (qRTPCR), TaqMan, gene expression micro arrays, Northern or Western blot procedures to measure gene expression and transcription.
  • qRTPCR quantitative Reverse transcriptase PCR
  • TaqMan gene expression micro arrays
  • Northern or Western blot procedures to measure gene expression and transcription.
  • QF-PCR quantitative fluorescent PCR
  • MF-PCR multiplex fluorescent PCR
  • RT-PCR real time PCR
  • PCR-RFLP restriction fragment length PCR
  • PCR-RFLP PCR-RFLP RT-PCR-RFLP
  • hot start PCR nested PCR, in situ polonony PCR, in situ rolling circle amplification, bridge PCR, picotiter PCR, emulsion PCR, next generation/massively parallel sequencing and single molecule sequencing.
  • amplification methods include selective amplification of target polynucleotide sequences, consensus sequence primed PCR (CP-PCR), arbitrarily primed PCR (AP-PCR), degenerate oligonucleotides-primed PCR (DOP-PCR) and nucleic acid based sequence amplification (NABS A).
  • CP-PCR consensus sequence primed PCR
  • AP-PCR arbitrarily primed PCR
  • DOP-PCR degenerate oligonucleotides-primed PCR
  • NABS A nucleic acid based sequence amplification
  • DNA methylation status can be determined in any number of ways such as by electrophoresis which includes capillary, capillary zone, capillary isoelectric focusing and capillary gel electrophoresis as well as capillary electrochromatography, micellar electrokinetic capillary chromatography and transient isotachophoresis by use of arrays, beads, gas chromatography, supercritical fluid chromatography, liquid chromatography (which encompasses partition, adsorption, ion
  • pathological condition or “disease condition” includes an abnormal malignancy as defined by objective or subjective manifestations of cancer diseases.
  • the assay of the present disclosure enables a molecular (i.e. genetic or epigenetic) determination to be made to complement other symptom-based diagnoses such as based on physiological or medical studies or may be made in its own right.
  • the assay may be part of a suite of diagnostic or prognostic genetic assays of embryos, pre- and postnatal subjects.
  • the terms "method”, “assay”, “system”, “test”, “determination”, “prognostic”, “diagnostic”, “report” and the like may all be used to describe an epigenetic assay including a methylation assay of selected regions of one or more loci listed in Table 2, the number selected on the basis of the required level of confidence.
  • the methylation assay determines the epigenetic profile or extent of methylation change compared to a control which suggests or indicates or is instructive of a cancerous condition.
  • the present disclosure also teaches epidemiological studies of populations including studies of different ethnic populations with cancer.
  • the present disclosure further provides a method of identifying an epigenetic profile in a population of subjects indicative of a cancerous pathological condition the method comprising screening for a change relative to a control in a statistically significant number of subjects of the extent of epigenetic change within or proximal to one or more loci listed in Table 2 or 3 wherein the extent of epigenetic change is indicative of the presence of the pathological condition or a propensity to develop same.
  • Also taught herein is a method of identifying a methylation profile in a population of subjects indicative of a cancerous pathological condition the method comprising screening for a change relative to a control in a statistically significant number of subjects the extent of methylation within or proximal to one or more loci listed in Table 2 or 3 wherein an increase or decrease in extent of methylation is indicative of the presence of the pathological condition or a propensity to develop same.
  • Another aspect of the present disclosure enabled herein is a method of identifying epigenetic profile in a population of subjects indicative of a cancerous pathological condition the method comprising screening for a change relative to a control in a statistically significant number of subjects the extent of epigenetic change within or proximal to from 1 to about 16 loci listed in Table 2 or 3 or in 1 to 16 regions within one or more loci listed in Table 2 or 3 wherein an elevation or decrease in extent of epigenetic change is indicative of the presence of the pathological condition or a propensity to develop same.
  • a further taught herein is a method of identifying a methylation profile in a population of subjects indicative of a cancerous pathological condition the method comprising screening for a change relative to a control in a statistically significant number of subjects the extent of methylation within or proximal to one or more loci listed in Table 2 or in 1 to 16 regions within one or more loci listed in Table 2 or 3 wherein an increase or decrease in extent of methylation is indicative of the presence of the pathological condition or a propensity to develop same.
  • the total number of sites selected depends on the level of confidence required for a diagnosis. For example, from about 40% to 100% confidence levels. A total of 16 different loci, or 16 different regions within one or more loci provides a 100% confidence level of the presence or absence of cancer.
  • proximal includes the region up to approximately lOOOkb upstream or downstream (5' or 3' terminal regions) of a locus.
  • a genome wide methylation map has been constructed in accordance with the present disclosure using standard techniques such as and DNA hybridisation microarray and high throughput mass spectrometry of various cells. Any cell type cell may be assayed. These cells include blood cells, CSF cells, bone marrow cells, buccal cells and cells from rectal swabs or faeces.
  • the present disclosure contemplates that the extent of epigenetic change in within or proximal to one or more loci listed in Table 2 or 3 which corresponds to a healthy condition or a level of disease within the spectrum of cancer.
  • the extent of change in epigenetic profile permits the sensitive determination of MRD in a subject and its use in prognosis of cancer.
  • the cancerous condition includes leukemias and its various types and sub-types such as pediatric and adult leukemias.
  • methylation may occur anywhere within the DNA or RNA of or proximal to a locus including of any cytosine whether in islands or shores or other areas of the nucleic acid.
  • cytosine or “C”, “CpG islands”, “CpNpG islands”, “island shores” and “shores” all include these basis in a locus or in a region up to approximately lOOOkb in distance upstream or downstream from a locus listed in Table 2 or 3 or more particularly Table 2 or 3 as well as within the locus and upstream and downstream enhancer elements. Multiple loci may be screened or multiple regions within one or more loci screened.
  • the terms "subject”, “case”, “patient”, “individual”, “target” and the like refer to any organism or cell of the organism on which an assay described herein is performed whether for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Typical subjects include both male and female humans but the present disclosure extends to experimental animals such as non-human mammals, (e.g., monkeys, chimpanzees, orangutangs, gorillas, mice, rats, rabbits, sheep, pigs, cows, horses and guinea pigs hamsters). Other test cells may be used such as yeast, microorganisms, single cell organisms to test fundatmental gene function which is conserved throughout phyla.
  • non-human mammals e.g., monkeys, chimpanzees, orangutangs, gorillas, mice, rats, rabbits, sheep, pigs, cows, horses and guinea pigs hamsters.
  • Other test cells may be used such as yeast, microorganisms,
  • the "subject” may also be referred to as a population since the present disclosure is useful in epidemiological studies or assays of an ethnic population.
  • genomic DNA includes all DNA in a cell, group of cells, or in an organelle of a cell and includes exogenous DNA such a transgenes introduced into a cell.
  • the present disclosure teaches a method for identifying an epigenetic profile in the genome of a cell indicative of a cancerous pathological condition, the method comprising screening for a change relative to the control in the extent of epigenetic change within or proximal to one or more loci listed in Table 2 or 3 or in 1 to 16 regions within one or more loci listed in Table 2 or 3 wherein the extent of epigenetic change is indicative of the presence of the cancerous pathological condition or a propensit to develop same.
  • This method is particularly useful in the screening of a change in extent of methylation or distribution of methylation sites.
  • the change may be indicative of health, remission or cancer.
  • loci may be grouped based on function, i.e. loci associated with (i) cell fate commitment; (ii) transcription factor activity; (iii) DNA binding; (iv) subcellular location; and (v) transcription regulator activity.
  • Any methylation assay may be employed such as bisulfite sequencing (see, for example, WO 2005/038051), methylation specific melting curve analysis (MS-MCA), high resolution melting (MS-HRM) [Dahl et al, Clin Chem JJ (4:790-793, 2007; Wojdacz et al, Nucleic Acids Res. 35(6)x4l, 2007], MALDI-TOF MS (Tost et al, Nucleic Acids Res 31(9):e5Q, 2003), methylation specific MLPA (Nygren et al, Nucleic Acids Res.
  • MS-MCA methylation specific melting curve analysis
  • MS-HRM high resolution melting
  • methylated-DNA precipitation/enrichment and methylation-sensitive restriction enzymes [Yegnasubramaniah et al, Nucleic Acids Res. 34(3)x ⁇ 9, 2006] or methylation sensitive oligonucleotide microarray (Gitan et al, Genome Res. 72(7;: 158-164, 2002), Infinium (Bibikova et al, Genome Research 16:383-393, 2006) and MethylLight (Trinh et al, 2001 supra; Shivapunkar et al, 2005 supra; WO 00/70090; US Patent No.
  • Amplification methodologies contemplated herein include the polymerase chain reaction (PCR) such as disclosed in U.S. Patent Nos. 4,683,202 and 4,683,195; the ligase chain reaction (LCR) such as disclosed in European Patent Application No. EP-A-320 308 and gap filling LCR (GLCR) or variations thereof such as disclosed in .
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • GLCR gap filling LCR
  • Other amplification techniques include Qp replicase such as described in the literature; Stand Displacement Amplification (SDA) such as described in European Patent Application Nos.
  • EP-A-497 272 and EP-A-500 224 ; Self-Sustained Sequence Replication (3SR) such as described in Fahy et ah, PCR Methods Appl. 1(1):25- 33, 1991) and Nucleic Acid Sequence-Based Amplification (NASBA) such as described in the literature.
  • 3SR Self-Sustained Sequence Replication
  • NASBA Nucleic Acid Sequence-Based Amplification
  • a PCR amplification process is useful in the practice of the assay enabled herein.
  • Another aspect of the present disclosure contemplates a method for determining the epigenetic profile within the genome of a eukaryotic cell or group of cells, the method comprising obtaining a sample of genomic DNA or RNA from the cell or group of cells and subjecting the genomic DNA or RNA to bisulfite treatment/conversion followed by primerrspecific amplification within one or more loci listed in Table 2 or 3 or in 1 to 16 regions within one or more loci listed in Table 2 or 3 and assaying for extent of change in epigenetic profile relative to a control.
  • the present disclosure further provides a method for determining the methylation profile within the genome of a eukaryotic cell or group of cells, the method comprising obtaining a sample of genomic DNA or RNA from the cell or group of cells and subjecting the genomic DNA or RNA to bisulfite treatment/conversion followed by primer-specific amplification within one or more loci listed in Table 2 or 3 or in 1 to 16 regions within one or more loci listed in Table 2 or 3 and assaying for extent of change in methylation relative to a control.
  • the present disclosure still further enables a method for determining the epigenetic profile within the genome of a eukaryotic cell or group of cells, the method comprising obtaining a sample of genomic DNA or RNA from the cell or group of cells and subjecting the genomic DNA or RNA to bisulfite treatment/conversion followed by primer-specific amplification within from 1 to 16 loci listed in Table 2 or 3 or in 1 to 16 regions within one or more loci listed in Table 2 or 3 and assaying for extent of change in epigenetic profile.
  • Yet a further aspect taught herein is a method for determining the methylation profile within the genome of a eukaryotic cell or group of cells, the method comprising obtaining a sample of genomic DNA or RNA from the cell or group of cells and subjecting the genomic DNA or RNA to bisulfite treatment/conversion followed by primer-specific amplification within from 1 to 16 loci listed in Table 2 or 3 or in 1 to 16 regions within one or more loci listed in Table 2 or 3 and assaying for extent of methylation relative to a control.
  • From 1 to 16 different loci may be assayed or from 1 to 16 different sites within from 1 to 16 loci may be assayed.
  • a change in epigenetic profile such as a change in extent of methylation or distribution of methylation sites compared to a control (i.e. a subject with known disease status) determines if a subject has cancer, is healthy or is in remission.
  • nucleic acid is a covalently linked sequence of nucleotides in which the 3' position of the phosphorylated pentose of one nucleotide is joined by a phosphodiester group to the 5' position of the pentose of the next nucleotide and in which the nucleotide residues are linked in specific sequence; i.e. a linear order of nucleotides.
  • a "polynucleotide” as used herein, is a nucleic acid containing a sequence that is greater than about 100 nucleotides in length.
  • An "oligonucleotide” as used herein, is a short polynucleotide or a portion of a polynucleotide.
  • An oligonucleotide typically contains a sequence of about two to about one hundred bases.
  • the word “oligo” .is sometimes used in place of the word “oligonucleotide”.
  • the term “oligo” also includes a particularly useful primer length in the practice of the present invention of up to about 10 nucleotides.
  • primer refers to an oligonucleotide or polynucleotide that is capable of hybridizing to another nucleic acid of interest under particular stringency conditions.
  • a primer may occur naturally as in a purified restriction digest or be produced synthetically, by recombinant means or by PCR amplification.
  • probe and “primers” may be used interchangeably, although to the extent that an oligonucleotide is used in a PCR or other amplification reaction, the term is generally "primer".
  • the ability to hybridize is dependent in part on the degree of complementarity between the nucleotide sequence of the primer and complementary sequence on the target DNA.
  • complementarity are used in reference to nucleic acids (i.e. a sequence of nucleotides) related by the well-known base-pairing rules that A pairs with T or U and C pairs with G.
  • sequence 5'-A-G-T-3' is complementary to the sequence 3'-T-C-A-5 * in DNA and 3'-U-C-A-5' in RNA or bisulfite treated genomic DNA.
  • Complementarity can be "partial" in which only some of the nucleotide bases are matched according to the base pairing rules.
  • nucleic acid strands there may be “complete” or “total” complementarity between the nucleic acid strands when all of the bases are matched according to base-pairing rules.
  • the degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands as known well in the art. This is of particular importance in detection methods that depend upon binding between nucleic acids, such as those of the invention.
  • substantially complementary is used to describe any primer that can hybridize to either or both strands of the target nucleic acid sequence under conditions of low stringency as described below or, preferably, in polymerase reaction buffer heated to 95°C and then cooled to room temperature.
  • the primer when the primer is referred to as partially or totally complementary to the target nucleic acid, that refers to the 3'-terminal region of the probe (i.e. within about 10 nucleotides of the 3'- terminal nucleotide position).
  • the present disclosure teaches, therefore, a methylation profile of the sites within one or more loci listed in Table 2 or 3 in a genome of a eukaryotic cell or group of cells, the methylation profile comprising the extent or level of methylation, the method comprising obtaining a sample of genomic DNA or RNA from the cell or group of cells, subjecting the restriction digested DNA or RNA or bisulfite treated DNA or RNA to an amplification reaction using primers selected to amplify one or more regions in one or more loci listed in Table 2 or 3 or modified primers which can amplify bisulfite treated DNA or RNA and then subjecting the amplified DNA or RNA to methylation detection means to determine relative to control the extent of methylation wherein a change in methylation relative to the control is indicative of a cancerous pathological condition.
  • the control may be a normal subject or a subject with known disease status.
  • kits for determining the epigenetic profile of one or more nucleotides at one or more sites within the genome of a eukaryotic cell or group of cells may comprise many different forms but in one embodiment, the kits comprise reagents for the bisulfite methylation assay and primers for amplification.
  • a further embodiment taught by the present disclosure is a kit for the use in the above methods comprising primers to amplify a particular site within one or more loci listed in Table 2 or 3 or in 1 to 16 regions within one or more loci listed in Table 2 or 3 or which amplify a strand after bisulfite conversion.
  • kits may also comprise instructions for use.
  • the kits are adapted to contain compartments for two or more of the above-listed components. Furthermore, buffers, nucleotides and/or enzymes may be combined into a single compartment.
  • instructions optionally present in such N kits instruct the user on how to use the components of the kit to perform the various methods taught herein. It is contemplated that these instructions include a description of the detection methods enabled herein.
  • kits which contain a primer for a nucleic acid target of interest with the primer being complementary to a predetermined nucleic acid target or a bisulfite treated template.
  • the kit contains multiple primers or probes, each of which contains a different base at an interrogation position or which is designed to interrogate different target DNA or RNA sequences.
  • multiple probes are provided for a set of nucleic acid target sequences that give rise to analytical results which are distinguishable for the various probes.
  • the multiple probes may be in microarray format for ease of use.
  • a kit comprises a vessel containing a purified and isolated enzyme whose activity is to release one or more nucleotides from the 3' terminus of a hybridized nucleic acid probe and a vessel containing pyrophosphate. In one embodiment, these items are combined in a single vessel. It is contemplated that the enzyme is either in solution or provided as a solid (e.g. as a lyophilized powder); the same is true for the pyrophosphate. Preferably, the enzyme is provided in solution. Some contemplated kits contain labeled nucleic acid probes. Other contemplated kits further comprise vessels containing labels and vessels containing reagents for attaching the labels.
  • Microtiter trays are particularly useful and these may comprise from two to 100,000 wells or from about six to about 10,000 wells or from about six to about 1 ,000 wells.
  • the present disclosure also teaches genome wide screening for epigenetic profiles including methylation profiles.
  • Another important application is in the high throughput screening of agents which are capable of demethylation genomes. This may be important, for example, in dedifferentiating cells and cancer therapies.
  • the present disclosure further enables a method for screening for an agent which modulates epigenetic regulation of one or more loci or a region within one or more loci listed in Table 2 or 3 or in 1 to 16 regions within one or more loci listed in Table 2 or 3 the method comprising screening for a change relative to a control in the epigenetic profile within or proximal to a locus in the presence or absence of an agent to be tested, wherein an agent is selected if it induces a change in the extent of epigenetic.
  • a method for screening for an agent which modulates methylation of one or more loci listed in Table 2 or 3 or in 1 to 16 regions within one or more loci listed in Table 2 or 3 comprising screening for a change relative to a control in the extent of methylation within or proximal to a locus in the presence or absence of an agent to be tested, wherein an agent is selected if it induces a change in the extent of methylation.
  • a further method for screening for an agent which modulates epigenesis of one or more loci listed in Table 2 or 3 or in 1 to 16 regions within one or more loci listed in Table 2 or 3 the method comprising screening for a change relative to a control in the extent of change in the epigenetic profile within or proximal to a locus in the presence or absence of an agent to be tested, wherein an agent is selected if it induces a change in the extent of epigenesis.
  • a still further method is provided for screening for an agent which modulates methylation of one or more loci listed in Table 2 or 3 or in 1 to 16 regions within one or more loci listed in Table 2 or 3 the method comprising screening for a change relative to a control in the extent of methylation within or proximal to a locus in the presence or absence of an agent to be tested, wherein an agent is selected if it induces a change in the extent of methylation.
  • the present disclosure further enables a method for monitoring the treatment of a cancer in which the treatment modulates the epigenetic profile of one or more loci or a region within one or more loci listed in Table 2 or 3 or in 1 to 16 regions within one or more loci listed in Table 2 or 3 the method comprising monitoring for a change relative to a control or a pre- and post-treatment sample in the epigenetic profile within or proximal to one or more loci.
  • a method for monitoring the treatment of a cancer in which the treatment modulates the methylation of one or more loci listed in Table 2 or 3 or in 1 to 16 regions within one or more loci listed in Table 2 or 3 the method comprising monitoring for a change relative to a control or a pre- and post-treatment sample in the extent of methylation within or proximal to one or more loci.
  • a further method for monitoring the treatment of a cancer is provided in which the treatment modulates the epigenetic profile of one or more loci listed in Table 2 or 3 or in 1 to 16 regions within one or more loci listed in Table 2 or 3 the method comprising monitoring for a change relative to a control or a pre- and post-treatment sample in the extent of the epigenetic profile within or proximal to one or more loci.
  • a still further method for monitoring the treatment of a cancer in which the treatment modulates the methylation of one or more loci or a region within one or more loci listed in Table 2 or 3 or in 1 to 16 regions within one or more loci listed in Table 2 or 3 the method comprising monitoring for a change relative to a control or a pre- and post- treatment sample in the extent of methylation within or proximal to one or more loci.
  • references to "one or more loci listed in Table 2 or 3" includes from 1 to 16 loci listed in Table 2 or 3.
  • the 1 to 16 loci may be associated with (i) cell fate commitment; (ii) transcription factor activity; (iii) DNA binding; (iv) subcellular location; and (v) transcription regulator activity (see Table 2).
  • the identification of MRD is provided such as a malignant cell at a frequency down to 10 " * to 10 "8 (i.e. one cell in from 10 4 to 10 8 cells) which may be instructive in the decision as to whether to apply more intensive therapy such as more toxic therapy, a bone marrow transplant or stem cell therapy.
  • methylation is detected in any C such as a C in CpG or CpNpG islands and/or island shores.
  • the cancer is a leukemia or its various types or sub-types.
  • RNA-encoding transgenes are transfected as a transgene into cells to methylate the gene, silence it and thereby correct the defect.
  • double stranded RNA-encoding transgenes are introduced with modulating sequences which protect it from methylation, keep it transcriptionally active and producing double stranded RNA.
  • the present disclosure further teaches a computer program and hardware which monitors the changing state, if any, of extent of methylation over time or in response to therapeutic and/or behavioral modification. This includes for monitoring MRD.
  • Such a computer program has important utility in monitoring disease progression, response to intervention and may guide modification of therapy or treatment.
  • the computer program is also useful in understanding the association between increasing methylation and disease progression.
  • index values are assigned to levels of methylation in selected loci or proximal thereto which are stored in a machine-readable storage medium, which is capable of processing the data to provide an extent of disease progression or change in methylation for a subject.
  • the disclosure teaches a computer program product for assessing progression of a pathological condition associated with cancer in a subject, the product comprising:
  • the present disclosure levels a computer for assessing an association between extent of methylation or other epigenetic change within one or more regions within one or more loci listed in Table 2 or 3 and progression of a cancerous condition wherein the computer comprises:
  • a machine-readable data storage medium comprising a data storage material encoded with machine-readable data, wherein the machine-readable data comprise values associated with extent of methylation or other epigenetic change in one or more regions of one or more loci listed in Table 2 or 3; (2) means to converting the value to a code; and
  • the present disclosure extends to a computer for assessing an association between extent of methylation or other epigenetic change within one or more loci listed in Table 2 or 3 or in 1 to 16 regions within one or more loci listed in Table 2 or 3 and progression of a cancer disease condition
  • the computer comprises a machine- readable data storage medium comprising a data storage material encoded within machine- readable data, wherein the machine-readable data comprise values associated with the features extent of methylation or other epigenetic change in one or more regions of one or more loci listed in Table 2 or 3.
  • the present disclosure also teaches a computer comprising: (1) a working memory for storing instructional codes for. processing the machine-readable data;
  • a central-processing unit coupled to the working memory and to the machine-readable data storage medium, for processing the machine-readable data to provide data instructional or informative of changing methylation or other epigenetic patterns or disease progression;
  • the computer system herein may also be linked to detection systems such as MALDI-TOF machines.
  • Reference to one or more loci herein includes from 1 to 100 such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100.
  • 1 to 100 such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
  • from 1 to 50 or 1 to 30 or 1 to 20 loci are screened including 1 to 16 such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16.
  • the present disclosure further encompasses from 1 to 100 sites or regions in 1 or more loci such as 16 sites from one or more loci.
  • the AROC is the chance of the classifer correctly ranking samples, i.e. the chance that the model would take a random sample from the positive class and a random sample from the negative class and rank the positive class higher than the negative class. This metric is popular in machine learning and biomedical research.
  • the AROC can be estimated as:
  • Genomic DNA was subjected to bisulphite conversion using either MethylEasy Xceed (Human Genetic Signatures) or EZ-96 DNA Methylation gold (Zymo Research) kits according to manufacturer's instructions. . Converted DNA was eluted with sufficient volume of elution buffer to give a final sample concentration of 20ng ⁇ L.
  • PCR amplification of bisulphite converted genomic DNA was performed using gene specific primers containing the necessary tags for SEQUENOM analysis (Table 7) and Fast Start PCR master mix (Roche Applied Sciences) according to manufacturer's instructions. Typical PCR cycling conditions performed are: 95°C for 10 minutes denaturation followed by five cycles of 95°C for 10 seconds denaturation, 56°C annealing for 20 seconds, 72°C extension for two minutes and 40 cycles of 95°C for 10 seconds denaturation, 56°C annealing for 20 seconds, 72°C extension for 1.5 minutes.
  • SEQUENOM EpiTYPER chemistry consisting of SAP treatment, RNA transcription and cleavage prior to mass spectrophotometry analysis as outlined in the manufacturer's instructions.
  • Mass spectra were processed using EpiTYPER viewer software vl.0.5 (SEQUENOM Inc.) and cleaned using an in-house R-script to remove poor quality CpG units and samples.
  • Heatmaps of SEQUENOM data were also drawn using heatmap.2 of the gplots library (http://www.r-project.org ' ).
  • MOSC1 NM_022746.2 0.79 0.03 0.76 0.76 23.60
  • MOSC2 NM_017898.3 0.79 0.04 0.75 0.75 18.69
  • Tables 13a through 13e show genetic loci with greater than 90% differential methylation between leukemia and controls.
  • Table 13a lists the top 100 genes identified using the centroid model (P ID:8917796) to separate leukemia bone marrow from normal bone marrow, of which any combination of 16 genes can determine this 100% of the time;
  • Table 13b lists the top 100 genes identified using the Linear Models for Microarray Analysis (LIMMA) [De Hertogh et al, BMC Bioinformatics 77:17, 2020 (PMID 20064233)] model to separate leukemia bone marrow from normal bone marrow, of which any combination of 16 genes can determine this 100% of the time;
  • Table 13c lists the top 100 genes identified using the Support Vector Macchine (SVM) [Varewyck and Martens, IEEE Trans.
  • SVM Support Vector Macchine
  • NGFIC NGFIC
  • NGFI-C NGFIC
  • GROl GROa
  • LGIL2 LGIL2; FU10675;

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Hospice & Palliative Care (AREA)
  • Biophysics (AREA)
  • Oncology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne généralement une analyse destinée à la détermination de profils épigénétiques, y compris de profils épigénétiques associés à une condition pathologique. La présente invention concerne une analyse destinée à détecter des profils épigénétiques associés au cancer, y compris sa forme ou son type, son état et le statut de la maladie résiduelle imperceptible. L'analyse de l'invention permet en outre l'identification et la surveillance des formes et des sous-types de leucémie et autres malignités hématologiques. La présente invention concerne en outre des kits et des dosages de médicaments ainsi que des procédés de criblage d'agents qui modulent la méthylation.
PCT/AU2011/001161 2010-09-10 2011-09-08 Analyse destinée à la détection et à la surveillance du cancer Ceased WO2012031329A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AU2010904085A AU2010904085A0 (en) 2010-09-10 Assay for detection and monitoring of cancer
AU2010904085 2010-09-10

Publications (1)

Publication Number Publication Date
WO2012031329A1 true WO2012031329A1 (fr) 2012-03-15

Family

ID=45810023

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/AU2011/001161 Ceased WO2012031329A1 (fr) 2010-09-10 2011-09-08 Analyse destinée à la détection et à la surveillance du cancer

Country Status (1)

Country Link
WO (1) WO2012031329A1 (fr)

Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014056986A1 (fr) 2012-10-11 2014-04-17 Universitat De Barcelona Procédé de prédiction de l'évolution clinique d'un patient souffrant d'une leucémie lymphoïde chronique (cll)
JP2015526096A (ja) * 2012-08-31 2015-09-10 国防医学院Ｎａｔｉｏｎａｌ Ｄｅｆｅｎｓｅ Ｍｅｄｉｃａｌ Ｃｅｎｔｅｒ 癌のスクリーニング方法
CN105408494A (zh) * 2012-05-11 2016-03-16 独立行政法人国立癌症研究中心 预测肾细胞癌的预后的方法
WO2016115530A1 (fr) * 2015-01-18 2016-07-21 The Regents Of The University Of California Procédé et système pour déterminer l'état d'un cancer
EP2898100B1 (fr) 2012-09-20 2017-11-22 The Chinese University Of Hong Kong Détermination non invasive d'un méthylome du foetus ou d'une tumeur à partir du plasma
WO2018009707A1 (fr) * 2016-07-06 2018-01-11 Youhealth Biotech, Limited Marqueurs de méthylation spécifiques d'une tumeur solide et utilisations de ces marqueurs
JP2018138036A (ja) * 2018-04-17 2018-09-06 国防医学院Ｎａｔｉｏｎａｌ Ｄｅｆｅｎｓｅ Ｍｅｄｉｃａｌ Ｃｅｎｔｅｒ 癌のスクリーニング方法
CN109456968A (zh) * 2018-12-29 2019-03-12 上海奕谱生物科技有限公司 基于甲基化修饰的肿瘤标记物stamp-ep5
US10392666B2 (en) 2012-09-20 2019-08-27 The Chinese University Of Hong Kong Non-invasive determination of methylome of tumor from plasma
US10513739B2 (en) 2017-03-02 2019-12-24 Youhealth Oncotech, Limited Methylation markers for diagnosing hepatocellular carcinoma and lung cancer
CN111088350A (zh) * 2020-02-24 2020-05-01 内蒙古医科大学 Mt2基因启动子区甲基化用检测引物与检测方法以及应用
US10706957B2 (en) 2012-09-20 2020-07-07 The Chinese University Of Hong Kong Non-invasive determination of methylome of tumor from plasma
US11062789B2 (en) 2014-07-18 2021-07-13 The Chinese University Of Hong Kong Methylation pattern analysis of tissues in a DNA mixture
WO2022040306A1 (fr) * 2020-08-19 2022-02-24 Mayo Foundation For Medical Education And Research Détection de lymphome non hodgkinien
US11410750B2 (en) 2018-09-27 2022-08-09 Grail, Llc Methylation markers and targeted methylation probe panel
US11435339B2 (en) 2016-11-30 2022-09-06 The Chinese University Of Hong Kong Analysis of cell-free DNA in urine
CN115341029A (zh) * 2022-04-18 2022-11-15 人和未来生物科技（长沙）有限公司 一种nell1基因甲基化检测试剂及其应用
US11514289B1 (en) 2016-03-09 2022-11-29 Freenome Holdings, Inc. Generating machine learning models using genetic data
US12024750B2 (en) 2018-04-02 2024-07-02 Grail, Llc Methylation markers and targeted methylation probe panel
US12027237B2 (en) 2018-03-13 2024-07-02 Grail, Llc Anomalous fragment detection and classification
US12234514B2 (en) 2018-12-21 2025-02-25 Grail, Inc. Source of origin deconvolution based on methylation fragments in cell-free DNA samples

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090005268A1 (en) * 2005-07-18 2009-01-01 Epigenomics Ag Compositions and Methods for Cancer Diagnostics Comprising Pan-Cancer Markers
WO2010086389A1 (fr) * 2009-01-28 2010-08-05 Ait Austrian Institute Of Technology Gmbh Essai de méthylation

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090005268A1 (en) * 2005-07-18 2009-01-01 Epigenomics Ag Compositions and Methods for Cancer Diagnostics Comprising Pan-Cancer Markers
WO2010086389A1 (fr) * 2009-01-28 2010-08-05 Ait Austrian Institute Of Technology Gmbh Essai de méthylation
WO2010086388A1 (fr) * 2009-01-28 2010-08-05 Ait Austrian Institute Of Technology Gmbh Marqueurs de méthylation pour le cancer du poumon

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ADORJAN, P. ET AL.: "Tumour class prediction and discovery by microarray-based DNA methylation analysis", NUCLEIC ACID RESEARCH, vol. 30, 2002, pages E21 *
MILANI, L. ET AL.: "DNA methylation for subtype classification and prediction of treatment outcome in patients with childhood acute lymphoblastic leukemia", BLOOD, vol. 115, 2010, pages 1214 - 1225, XP002731851, DOI: doi:10.1182/BLOOD-2009-04-214668 *

Cited By (53)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105408494B (zh) * 2012-05-11 2018-10-16 独立行政法人国立癌症研究中心 预测肾细胞癌的预后的方法
CN105408494A (zh) * 2012-05-11 2016-03-16 独立行政法人国立癌症研究中心 预测肾细胞癌的预后的方法
EP2848697A4 (fr) * 2012-05-11 2016-05-04 Nat Cancer Ct Procédé pour la prédiction du pronostic d'un néphrocarcinome
JP2015526096A (ja) * 2012-08-31 2015-09-10 国防医学院Ｎａｔｉｏｎａｌ Ｄｅｆｅｎｓｅ Ｍｅｄｉｃａｌ Ｃｅｎｔｅｒ 癌のスクリーニング方法
US10221458B2 (en) 2012-08-31 2019-03-05 National Defense Medical Center Method for screening cancer
EP2891720A4 (fr) * 2012-08-31 2016-11-09 Nat Defense Medical Ct Méthode de dépistage du cancer
JP2024009833A (ja) * 2012-09-20 2024-01-23 ザ チャイニーズ ユニバーシティ オブ ホンコン 血漿による胎児または腫瘍のメチロームの非侵襲的決定
EP4335929A3 (fr) * 2012-09-20 2024-05-29 The Chinese University of Hong Kong Détermination non invasive du méthylome d'une tumeur à partir du plasma
EP3536807B1 (fr) * 2012-09-20 2023-11-29 The Chinese University of Hong Kong Détermination non invasive d'un méthylome d'une tumeur à partir du plasma
EP3839065A1 (fr) * 2012-09-20 2021-06-23 The Chinese University Of Hong Kong Détermination non invasive d'un méthylome d'une tumeur à partir du plasma
EP3354747B1 (fr) 2012-09-20 2021-02-17 The Chinese University Of Hong Kong Détermination non invasive d'un méthylome d'une tumeur à partir du plasma
EP2898100B1 (fr) 2012-09-20 2017-11-22 The Chinese University Of Hong Kong Détermination non invasive d'un méthylome du foetus ou d'une tumeur à partir du plasma
IL268512B2 (en) * 2012-09-20 2023-06-01 Univ Hong Kong Chinese Non-invasive determination of fetal or tumor methylation from plasma
AU2020200128B2 (en) * 2012-09-20 2021-11-18 The Chinese University Of Hong Kong Non-invasive determination of methylome of fetus or tumor from plasma
US10392666B2 (en) 2012-09-20 2019-08-27 The Chinese University Of Hong Kong Non-invasive determination of methylome of tumor from plasma
EP2898100B2 (fr) † 2012-09-20 2023-05-10 The Chinese University Of Hong Kong Détermination non invasive d'un méthylome du foetus ou d'une tumeur à partir du plasma
US11274347B2 (en) 2012-09-20 2022-03-15 The Chinese University Of Hong Kong Non-invasive determination of type of cancer
JP2022031753A (ja) * 2012-09-20 2022-02-22 ザ チャイニーズ ユニバーシティ オブ ホンコン 血漿による胎児または腫瘍のメチロームの非侵襲的決定
JP7594817B2 (ja) 2012-09-20 2024-12-05 ザ チャイニーズ ユニバーシティ オブ ホンコン 血漿による胎児または腫瘍のメチロームの非侵襲的決定
US10706957B2 (en) 2012-09-20 2020-07-07 The Chinese University Of Hong Kong Non-invasive determination of methylome of tumor from plasma
WO2014056986A1 (fr) 2012-10-11 2014-04-17 Universitat De Barcelona Procédé de prédiction de l'évolution clinique d'un patient souffrant d'une leucémie lymphoïde chronique (cll)
US11984195B2 (en) 2014-07-18 2024-05-14 The Chinese University Of Hong Kong Methylation pattern analysis of tissues in a DNA mixture
US11062789B2 (en) 2014-07-18 2021-07-13 The Chinese University Of Hong Kong Methylation pattern analysis of tissues in a DNA mixture
US9984201B2 (en) 2015-01-18 2018-05-29 Youhealth Biotech, Limited Method and system for determining cancer status
EA036566B1 (ru) * 2015-01-18 2020-11-24 Зе Реджентс Оф Зе Юниверсити Оф Калифорния Способ и система определения статуса злокачественной опухоли
WO2016115530A1 (fr) * 2015-01-18 2016-07-21 The Regents Of The University Of California Procédé et système pour déterminer l'état d'un cancer
US11514289B1 (en) 2016-03-09 2022-11-29 Freenome Holdings, Inc. Generating machine learning models using genetic data
US12242943B2 (en) 2016-03-09 2025-03-04 Freenome Holdings, Inc. Generating machine learning models using genetic data
US10544467B2 (en) 2016-07-06 2020-01-28 Youhealth Oncotech, Limited Solid tumor methylation markers and uses thereof
WO2018009707A1 (fr) * 2016-07-06 2018-01-11 Youhealth Biotech, Limited Marqueurs de méthylation spécifiques d'une tumeur solide et utilisations de ces marqueurs
CN107847515A (zh) * 2016-07-06 2018-03-27 优美佳生物技术有限公司 实体瘤甲基化标志物及其用途
US11435339B2 (en) 2016-11-30 2022-09-06 The Chinese University Of Hong Kong Analysis of cell-free DNA in urine
US10513739B2 (en) 2017-03-02 2019-12-24 Youhealth Oncotech, Limited Methylation markers for diagnosing hepatocellular carcinoma and lung cancer
US12027237B2 (en) 2018-03-13 2024-07-02 Grail, Llc Anomalous fragment detection and classification
US12024750B2 (en) 2018-04-02 2024-07-02 Grail, Llc Methylation markers and targeted methylation probe panel
US12435375B2 (en) 2018-04-02 2025-10-07 Grail, Inc. Methylation markers and targeted methylation probe panel
JP2018138036A (ja) * 2018-04-17 2018-09-06 国防医学院Ｎａｔｉｏｎａｌ Ｄｅｆｅｎｓｅ Ｍｅｄｉｃａｌ Ｃｅｎｔｅｒ 癌のスクリーニング方法
US11795513B2 (en) 2018-09-27 2023-10-24 Grail, Llc Methylation markers and targeted methylation probe panel
US11410750B2 (en) 2018-09-27 2022-08-09 Grail, Llc Methylation markers and targeted methylation probe panel
US12410482B2 (en) 2018-09-27 2025-09-09 Grail, Inc. Methylation markers and targeted methylation probe panel
US11685958B2 (en) 2018-09-27 2023-06-27 Grail, Llc Methylation markers and targeted methylation probe panel
US11725251B2 (en) 2018-09-27 2023-08-15 Grail, Llc Methylation markers and targeted methylation probe panel
US12234514B2 (en) 2018-12-21 2025-02-25 Grail, Inc. Source of origin deconvolution based on methylation fragments in cell-free DNA samples
JP2022515666A (ja) * 2018-12-29 2022-02-21 上▲海▼奕▲譜▼生物科技有限公司 メチル化修飾に基づく腫瘍マーカーｓｔａｍｐ－ｅｐ５
CN109456968B (zh) * 2018-12-29 2022-10-04 上海奕谱生物科技有限公司 基于甲基化修饰的肿瘤标记物
US20220195528A1 (en) * 2018-12-29 2022-06-23 Shanghai Epiprobe Biotechnology Co., Ltd. Tumor marker stamp-ep5 based on methylated modification
JP7407824B2 (ja) 2018-12-29 2024-01-04 上▲海▼奕▲譜▼生物科技有限公司 メチル化修飾に基づく腫瘍マーカーｓｔａｍｐ－ｅｐ５
WO2020135862A1 (fr) * 2018-12-29 2020-07-02 上海奕谱生物科技有限公司 Marqueur tumoral stamp-ep5 basé sur une modification méthylée
CN109456968A (zh) * 2018-12-29 2019-03-12 上海奕谱生物科技有限公司 基于甲基化修饰的肿瘤标记物stamp-ep5
CN111088350A (zh) * 2020-02-24 2020-05-01 内蒙古医科大学 Mt2基因启动子区甲基化用检测引物与检测方法以及应用
WO2022040306A1 (fr) * 2020-08-19 2022-02-24 Mayo Foundation For Medical Education And Research Détection de lymphome non hodgkinien
JP2023539128A (ja) * 2020-08-19 2023-09-13 マヨ ファウンデーション フォア メディカル エデュケーション アンド リサーチ 非ホジキンリンパ腫の検出
CN115341029A (zh) * 2022-04-18 2022-11-15 人和未来生物科技（长沙）有限公司 一种nell1基因甲基化检测试剂及其应用

Similar Documents

Publication Publication Date Title
WO2012031329A1 (fr) Analyse destinée à la détection et à la surveillance du cancer
JP7757338B2 (ja) 乳癌の検出
AU2022291502B2 (en) Detecting hepatocellular carcinoma
US12442043B2 (en) Detecting ovarian cancer
US11384401B2 (en) Detecting gastrointestinal neoplasms
AU2017251832B2 (en) Non-invasive determination of methylome of fetus or tumor from plasma
Sandoval et al. Validation of a DNA methylation microarray for 450,000 CpG sites in the human genome
Dehan et al. DNA methylation and cancer diagnosis: new methods and applications
US9957570B2 (en) DNA hypermethylation diagnostic biomarkers for colorectal cancer
Kron et al. Discovery of novel hypermethylated genes in prostate cancer using genomic CpG island microarrays
KR20210146983A (ko) 혈장에서 췌장 관상 선암종의 검출
WO2009108917A2 (fr) Marqueurs pour la détection améliorée du cancer du sein
US20230242995A1 (en) Method for detecting colorectal cancer
US20220220560A1 (en) Tumor marker stamp-ep3 based on methylation modification
EP2707506A2 (fr) Procédé de détection d'un cancer par l'intermédiaire d'une perte généralisée de stabilité de domaines épigénétiques, et compositions associées
JP2024020392A (ja) 特定の遺伝子のｃｐｇメチル化変化を利用した肝癌診断用組成物およびその使用
US20220195528A1 (en) Tumor marker stamp-ep5 based on methylated modification
WO2020254405A1 (fr) Prédiction de l'âge à l'aide de signatures de méthylation de l'adn
TW202417642A (zh) 鑑別癌症的甲基化標誌物及應用
Lee et al. Hypermethylation of PDX1, EN2, and MSX1 predicts the prognosis of colorectal cancer
Novak et al. Cell-type specific DNA methylation patterns define human breast cellular identity
Marsit et al. Blood-derived DNA methylation markers of cancer risk
US20220177973A1 (en) Methylation modification-based tumor marker stamp-ep6
CN102016067A (zh) 前列腺癌中gstp1高甲基化的检测
EP3964578A1 (fr) Marqueur tumoral stamp-ep8 basé sur une modification de méthylation et son application

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11822937

Country of ref document: EP

Kind code of ref document: A1

DPE1 Request for preliminary examination filed after expiration of 19th month from priority date (pct application filed from 20040101)
NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 11822937

Country of ref document: EP

Kind code of ref document: A1