WO2022139581A1 - Detection of nucleic acid markers in urine using dna methylation analysis - Google Patents
Detection of nucleic acid markers in urine using dna methylation analysis Download PDFInfo
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Classifications
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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
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- 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
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/154—Methylation markers
Definitions
- Urinary cfDNA can roughly be divided into two groups based on fragment size, comprising high- and low molecular weight (MW) groups.
- the high-MW group consists of heterogeneous DNA fragments of 1 kilobasepair (kbp) and larger. Without being bound by theory it is believed that such fragments originate from the cell debris of the urogenital tract 20.21 .
- the low -MW group consists of smaller DNA fragments of up to 250bp. Without being bound by theory it is believed that the low-MW fraction of urinary DNA is partially derived from the blood circulation, allowing detection of ctDNA in urine samples 21,22 .
- the invention provides a kit of parts according to daims 15-18, for use in a method of diagnosing colorectal cancer, colorectal metastasis, recurrent colorectal cancer or a combination thereof in a subject, the method comprising collecting nucleic add from a sample comprising at least 10 ml, preferably at least 20 ml of urine of said subject, processing the nudeic acid to isolate cell free nucleic add enriched for fragments of 150 nucleic add bases or less, and determining whether the enriched nucleic add comprises methylated nudeic acid of the SEPTIN-9 gene or a promoter region thereof and the SDC2 gene or a promoter region thereof
- DNA is isolated from the urine sample. More preferably cell free genomic DNA (cfDNA) is isolated.
- Methods for the isolation of DNA or cell free DNA from urine are well known in the art.
- the Quick-DNATM Urine Kit Zymo Research, Orange, CA, USA
- a DNA isolation method is used that is specifically tested for its applicability for isolation of DNA from urine.
- Various commercial DNA isolation kits designed and marketed for use with urine are available. In the present invention a method of the invention use such a commercial kit.
- the invention further provides a method of determining whether a human subject has colorectal cancer, a colorectal metastasis, a recurrent colorectal cancer or a combination thereof comprising collecting nucleic add from a sample comprising at least 10 ml, preferably at least 20 ml of urine of said subject, processing the nudeic add to isolate cell free nudeic add enriched for fragments of 150 nudeic add bases or less, and determining whether the enriched nucleic add comprises methylated nudeic acid of the SEPTIN-9 gene or a promoter region thereof and methylated nucleic add of the SDC2 gene or a promoter region thereof and determining that said subject has colorectal cancer, a colorectal metastasis, a recurrent colorectal cancer or a combination thereof based on the presence and/or the level of said methylated nudeic add.
- the cancer treatment is preferably tumor resection, chemotherapy, radiotherapy, immunotherapy or targeted therapy such as with an antibody such as Bevacuzimab.
- the cancer treatment can also include a treatment to prevent the development of cancer such as with adenoma.
- Adenoma and particularly advanced adenoma are often removed surgically via endoscopic intervention.
- the terms tumor and cancer are used interchangeably herein.
- Tumor resection indudes removal of cancer tissue or adenoma by surgery and removal of cancer tissue or adenoma via endoscopic intervention.
- Such a kit may comprise one or more of the following components: a container for collecting urine, a container filled with EDTA to achieve an EDTA concentration of at least 40 mM and/or one or more other additives, and test tubes for analysis.
- Said means for detection of DNA methylation may comprise primers and optionally a probe suitable for MSP or qMSP of the genes disclosed herein or a promoter region thereof, preferably primers as described herein, and/or methylation-sensitive restriction enzymes.
- said means comprise primers suitable for determining DNA methylation of a SEPTIN-9 promoter and SDC2 promoter.
- Said means for detection of DNA methylation may further comprise means for isolating DNA, preferably genomic DNA and/or bisulfite for converting isolated methylated DNA
- an element means one element or more than one element.
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- Engineering & Computer Science (AREA)
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Abstract
The invention relates to means and methods for detecting or determining the level of nucleic acid in urine of a human subject, the method comprising the steps of collecting nucleic acid from a sample of at least 1.0 ml, preferably at least 20 ml of urine of said subject, processing the sample to isolate cell free nucleic acid enriched for fragments of 150 nucleic acid bases or less, and determining whether the enriched nucleic acid comprises methylated nucleic acid of the SEPTIN-9 gene or a promoter region thereof and methylated nucleic acid of the SDC2 gene or a promoter region thereof. The means and methods may be used to arrive at a diagnosis of colorectal cancer, colorectal metastasis, recurrent colorectal cancer or a combination thereof in the human subject. The invention further relates to methods of treatment of human subjects with colorectal cancer, colorectal metastasis, recurrent colorectal cancer or a combination thereof comprising using the means and methods.
Description
Title: Detection of nucleic add markers in urine using DNA methylation analysis
FIELD OF THE INVENTION
The invention relates to the field of cancer diagnostics. In particular, the invention relates to the field of colon cancer detection. The invention further relates to detection methods of methylated nucleic add of the genes SEPTIN-9 and SDC2 or promoter regions thereof.
BACKGROUND OF THE INVENTION
Colorectal cancer (CRC) is the third most commonly diagnosed cancer worldwide and the second leading cause for cancer-related death, accounting for 10-12% of all cancer cases 1,2. Currently, there is a high clinical need for a non- invasive CRC biomarker test. Ideally the biomarker should have high accuracy and be able to provide means for detection of CRC, prognostication and disease monitoring during and after treatment s. Due to the absence of symptoms during early stages of disease, nearly half of patients are diagnosed at an advanced stage. Cancer stage at diagnosis however strongly correlates with survival, illustrated by the 5-year survival rate of 95% for stage I as compared to only 11% for stage IV 2. Timely detection of CRC could improve mortality rates due to better chances of diagnosing CRC at a more curable disease stage 4. Screening for CRC is therefore recommended in the western world as it is helps to lower the death rate due to CRC 5. Screening tests are stool-based or visual. However, both are cumbersome and colonoscopy carries risks as it is invasive 6,7. The ongoing development and increasing palette of CRC treatments require better tools to prognosticate patients. As molecular subtyping is key, colonoscopies and invasive biopsies are often warranted to decide on the appropriate treatment strategy 8,9. Furthermore, more accurate disease monitoring and detection of minimal residual disease following treatment is necessary. Existing tools, including imaging and engrained protein-based biomarker CEA, lack sensitivity or come with risks. Non-invasive means for accurate detection of a molecular signature of CRC would mean opportunities for each of the early detection, prognostication and monitoring modalities. This urgent need has led to extensive research on molecular CRC biomarkers in recent years, with growing interest in liquid biopsies.
A liquid biopsy' enables the analysis of cancer derived molecules in biofluids, including peripheral blood, saliva and urine 10. Blood containing cell free DNA (cfDNA) is the most studied liquid biopsy to date. Cell free DNA consists of extracellular nucleic acids released into the circulation by means of necrosis, apoptosis and active secretion n. A small fraction of the total cfDNA consists of tumor derived DNA, so called circulating tumor DNA (ctDNA). Detection of ctDNA can be facilitated by analysis of DNA methylation, which is the enzymatically induced covalent binding of a methyl group (-CHs) to cytosine-guanine dinucleotides (CpG) ,2. It is a common epigenetic regulator of gene expression, frequently altered in cancer 13. Increased methylation in CpG dense regions, called CpG islands and located in promoter regions of tumor suppressor genes, can lead to inactivation of tumor suppressor genes. As this is believed to be an early and critical event in CRC development,
analysis of ctDNA methylation has potential to serve as a biomarker for CRC3,14-16, Circulating tumor DNA is excreted in urine through glomerular filtration, which propagates urine analysis as a true non-invasive method of cancer detection 17- 19. Despite advances in ctDNA diagnostics in blood, the process of venipuncture remains invasive and many challenges still have to be tackled. Analysis of ctDNA in urine features a non-invasive and logistically attractive way of testing.
Urinary cfDNA can roughly be divided into two groups based on fragment size, comprising high- and low molecular weight (MW) groups. The high-MW group consists of heterogeneous DNA fragments of 1 kilobasepair (kbp) and larger. Without being bound by theory it is believed that such fragments originate from the cell debris of the urogenital tract 20.21. The low -MW group consists of smaller DNA fragments of up to 250bp. Without being bound by theory it is believed that the low-MW fraction of urinary DNA is partially derived from the blood circulation, allowing detection of ctDNA in urine samples 21,22. In the present invention urine samples were enriched for low-MW DNA by centrifugation, which partly separates potential tumor DNA from non-specific high-MW DNA 18. In the present invention it was found that, in contrast to for example bladder cancer, in which high-MW DNA present in urinary sediment is suitable for tumor DNA detection 23, the supernatant fraction is very suited for the detection of colon cancer, metastasis or recurrence thereof, in urine. In particular using methylation markers for the SEPTIN-9 and the SDC2 genes.
SUMMARY OF THE INVENTION
In one embodiment the invention provides a method of detecting the presence of nucleic acid in urine of a human subject, the method comprising the steps of collecting nucleic add from a sample of at least 10 ml, preferably at least 20 ml of urine of said subject, processing the sample to isolate cell free nucleic add enriched for fragments of 150 nucleic add bases or less, and determining whether the enriched nucleic add comprises methylated nucleic add of the SEPTIN-9 gene or a promoter region thereof and methylated nudeic add of the SDC2 gene or a promoter region thereof. Such methods may further comprise determining the level of said methylated SEPTIN-9 and SDC2 nudeic add in said enriched nudeic add.
In a preferred embodiment the method further comprises predicting from the presence and/or the level of said methylated SEPTIN-9 and SDC2 nudeic add whether said subject has colorectal cancer, a colorectal metastasis, a recurrent colorectal cancer or a combination thereof, or the risk of developing said cancer.
The invention also provides a method of providing a human subject in need thereof with a treatment of colorectal cancer, a colorectal metastasis, a recurrent colorectal cancer or a combination thereof the method comprising: providing a urine sample of at least 10 ml, preferably at least 20 ml of the subject, isolating cell free nucleic add enriched for DNA fragments of 150 nudeic acid bases or less from said urine; detecting the presence or the level of methylation of the SEPTIN-9 gene or a promoter region thereof and the SDC2 gene or a promoter region thereof in said enriched nudeic add, determining from the presence and or the level of said methylation that the human subject has colorectal cancer, a colorectal metastasis, a recurrent colorectal cancer or a combination thereof and providing
a treatment for said colorectal cancer, a colorectal metastasis, a recurrent colorectal cancer or a combination thereof to the subject in need thereof. The cancer treatment is preferably tumor resection, chemotherapy, radiotherapy, or an immunotherapy or targeted therapy such as but not limited to therapy that includes administration of the antibody Bevacuzimab. The cancer treatment can also include a treatment to prevent the development of cancer such as with adenoma. Adenoma and particularly advanced adenoma are often removed surgically via endoscopic intervention.
The invention further provides a method of determining whether a human subject has colorectal cancer, a colorectal metastasis, a recurrent colorectal cancer or a combination thereof comprising collecting nudeic add from a sample comprising at least 10 ml, preferably at least 20 ml of urine of said subject, processing the nudeic add to isolate cell free nudeic add enriched for fragments of 150 nucleic add bases or less, and determining whether the enriched nudeic add comprises methylated nudeic add of the SEPTIN-9 gene or a promoter region thereof and methylated nudeic add of the SDC2 gene or a promoter region thereof, and determining that said subject has colorectal cancer, a colorectal metastasis, a recurrent colorectal cancer or a combination thereof based on the presence and/or the level of said methylated nucleic add.
Detecting and/or determining the level of methylation may comprise a sequencing step, preferably a single molecule sequence step. Said detecting and/or determining the level of methylation preferably comprises performing a nucleic acid amplification step with said enriched nucleic acid. As a method to discriminate methylated nucleic add from unmethylated nucleic add it is preferred that said (enriched) nudeic add is treated with bisulfite prior to said detecting and/or determining, which preferably comprises performing a nucleic acid amplification. Said nucleic acid amplification step preferably comprises nucleic add amplification with a first and a second primer pair of which the primers of the first primer pair hybridize under stringent conditions to respectively the nucleic add sequence of SEQ ID NO: 1, or a complement thereof, and the nucleic acid sequence of SEQ ID NO: 2 or a complement thereof and the primers of the second primer pair hybridize under stringent conditions to respectively the nudeic acid sequence of SEQ ID NO: 4, or a complement thereof, and to the nudeic add sequence of SEQ ID NO: 5 or a complement thereof.
Cell free nucleic acid is preferably collected from the supernatant of centrifugated urine.
Determining the level of methylated nucleic acid of the SEPTIN-9 gene or a promoter region thereof and methylated nucleic acid of the SDC2 gene or a promoter region thereof preferably comprises comparing detected levels with a reference.
Nucleic acid amplified with the indicated primers is preferably probed with a first probe that hybridizes under stringent conditions to the nucleic acid sequence of SEQ ID NO: 3 and a second probe that hybridizes under stringent conditions to the nucleic acid sequence of SEQ ID NO: 6.
A urine sample preferably has been treated with a preservative, preferably ethylenediaminetetraacetic add (EDTA).
The invention also provides a kit of parts comprising means for the detection of DNA methylation of nudeic add of at most 10 genes or promoter regions thereof comprising wherein two of said genes or promoter regions thereof are SEPTIN-9 and SDC2.
The kit of parts preferably comprises a first and a second primer pair of which the primers of the first primer pair hybridize under stringent conditions to respectively the nucleic acid sequence of SEQ ID NO: 1, or a complement thereof and the nucleic acid sequence of SEQ ID NO: 2 or a complement thereof, and the primers of the second primer pair hybridize under stringent conditions to respectively the nucleic acid sequence of SEQ ID NO: 4, or a complement thereof, and to the nucleic add sequence of SEQ ID NO: 5 or a complement thereof. In one embodiment the kit comprises a first and a second probe wherein said first probe hybridizes under stringent conditions to the nucleic add sequence of SEQ ID NO: 3 and said second probe hybridizes under stringent conditions to the nucleic acid sequence of SEQ ID NO: 6. The kit preferably comprises a DNA isolation kit specifically designed to collect nucleic add from urine.
In one embodiment the invention provides a kit of parts according to daims 15-18, for use in a method of diagnosing colorectal cancer, colorectal metastasis, recurrent colorectal cancer or a combination thereof in a subject, the method comprising collecting nucleic add from a sample comprising at least 10 ml, preferably at least 20 ml of urine of said subject, processing the nudeic acid to isolate cell free nucleic add enriched for fragments of 150 nucleic add bases or less, and determining whether the enriched nucleic add comprises methylated nudeic acid of the SEPTIN-9 gene or a promoter region thereof and the SDC2 gene or a promoter region thereof
In one embodiment the urine has been collected from a pre-resection human subject. In other embodiments the urine has been collected from a human subject that has is undergoing treatment for colorectal cancer, a colorectal metastasis, a recurrent colorectal cancer or a combination thereof. Such urine samples are suited to follow the effect of the treatment or to monitor for reappearance of the cancer. In one embodiment the diagnosis returned is no colorectal cancer, colorectal metastasis, recurrent colorectal cancer or a combination thereof when the detection of methylated SEPTIN-9 nucleic add is below a predefined threshold or when a combined SEPTIN-9/SDC2 test is negative.
DETAILED DESCRIPTION
The SEPTIN-9 gene is known under a number of aliases some of which are Septin 9; Ov/Br Septin; MLL Septin-Like Fusion Protein MSF-A; Ovarian/Breast Septin; Septin Dl; KIAA099. Additional gene information for SEPTIN9 Gene are HGNC(7323); Entrez Gene(10801); Ensembl(ENSG00000184640); OMIM(604061); UniProtKB(Q9UHD8); Open Targets Platform(ENSG00000184640).
The SDC2 gene is known under a number of aliases such as Syndecan 2; Fibroglyean 2; SYND2; Heparan Sulfate Proteoglycan 1, Cell Surface-Associated; Heparan Sulfate Proteoglycan Core Protein; Syndecan Proteoglycan 2; Syndecan-2; CD362; HSPG1. Additional gene information for the SDC2 gene: HGNC(10659); Entrez Gene(6383); Ensembl(ENSG00000169439); OMIM(142460); UniProtKB(P34741); Open Targets Platform(ENSGOOOOO 169439).
The ACTB gene is known under a number of aliases such as Actin Beta; Actin Cytoplasmic 1; PS1TP5-Binding Protein 1; Beta Cytoskeletal Actin; I(2)-Actin; Beta-Actin; PS1TP5BP1; B-Actin. External Ids for ACTB Gene HGNC: 132; Entrez Gene: 60; Ensembl: ENSG00000075624; OMIM: 102630; UniProtKB: P60709. The ACTB gene is typically used as a reference.
The present inventors have identified the SEPTIN-9 and the SDC2 genes and/or promoter regions thereof that were significantly hypermethylated in urine samples of colorectal cancer, colorectal metastasis or recurrent colorectal cancer patients. The methods used in the examples comprise quantitative methylation specific polymerase chain reaction, particularly using multiplex PCR and a comparing with a non -related marker. Although this is a preferred embodiment, the invention is not limited to these particular methods. Various quantitative methylation specific analysis methods are known in the art. It was found among others that the use of a combination of the SEPTIN-9 and the SDC2 methylation markers in the urine, enables prediction of the subject having colorectal cancer, colorectal metastasis or recurrent colorectal cancer with particularly high specificity and sensitivity. In addition, contrary to known methods that use genetic markers for diagnosing colorectal cancer, colorectal metastasis or recurrent colorectal cancer, methods of the present invention allow the diagnosis of in principle any stage or grade of colorectal cancer, colorectal metastasis or recurrent colorectal cancer. This has the advantage that the same markers and thus the same test can be used for diagnosis or prognosis for all grades and stages of these cancers. It is no longer necessary to use different genetic markers or combinations of genetic marker to include all grades and stages.
A colorectal metastasis is a cancer in a location different from the site of the primary tumor in the colon which has originated from this primary tumor or an earlier metastasis thereof.
A “recurrent colorectal cancer” as used herein refers to forms of colorectal cancer that have reoccurred at the site of the primary tumor after an intervention, typically surgical intervention, to remove existing primary colorectal cancer.
A method of the invention comprises determining DNA methylation of nucleic acid of the SEPTIN-9 gene or promoter region thereof and DNA methylation of nucleic acid of the SDC2 gene or promoter region thereof. In particular DNA methylation of genomic DNA is determined.
Hypermethylation of the mentioned genes and/or promoter regions thereof indicates the individual is likely to have colorectal cancer, colorectal metastasis or recurrent colorectal
cancer. In some embodiments the hypermethylation indicates a risk of developing colorectal cancer, colorectal metastasis or recurrent colorectal cancer in the future. This can be because the detected stage is pre-eaneerous or too small to be detected using routine other means such a colonoscopy or non-specific scanning methods. One such pre-cancerous stage is advanced adenocarcinoma.
The term "hypermethylation" as used herein refers to any methylation of cytosine at a position that is normally unmethylated in the relevant gene sequences. Preferably, DNA methylation or hypermethylation of the promoter regions is detected. In a further preferred embodiment of the invention the DNA methylation or hypermethylation is detected in the CpG rich sequences in the promoter regions of the SEPTIN-9 and SDC2 genes. Suitable sources to identify the sequences of the genes are indicated herein above. These can also be used to identify sequences of the relevant promoter sequences. CpG elements therein can form the target of methylation. Multiple occurrences of CpG elements in a region are often referred to as CpG islands. Methylation of more or all of the CpG elements in such islands or of more closely associated islands is often referred to as hypermethylation. In the present invention methylation and hypermethylation are used interchangeably and have the same meaning, i.e. methylation of a CpG that is typically not methylated in nucleic acid of a “normal" colon cell.
The colorectal cancer, colorectal metastasis or recurrent colorectal cancer that is diagnosed, determined, typed, etc. with the methods of the invention is preferably a primary cancer. These can be particularly well detected. An advantage is that the present methods are particularly suited for general population monitoring studies. Human subjects with a positive test can be selected for further investigation and/or a conformation using an unrelated test.
A method of the invention is also well suited to monitor treatment of colorectal cancer, colorectal metastasis or recurrent colorectal cancer. It can monitor the impact of treatment that is given in parallel to the detection, or it can monitor the effectiveness of a remission or other result of treatment in the time after treatment has stopped. A positive result in a method can indicate recurrence of the tumor and/or a metastasis.
Currently there are no urine-based tests for colorectal cancer detection. Feces- and plasma- based tests do exist or are in development. The present inventors have found that the diagnostic performance of a combination of two hypermethylation markers as described herein, in particular the combination of SEPTIN-9 and SDC2, allowed detection of colorectal cancer, colorectal metastasis or recurrent colorectal cancer in urine with a sensitivity of 86-88% at a specificity of 67-70%.
It is preferred that DNA is isolated from the urine sample. More preferably cell free genomic DNA (cfDNA) is isolated. Methods for the isolation of DNA or cell free DNA from urine are well known in the art. As an example the Quick-DNA™ Urine Kit (Zymo Research, Orange, CA, USA) can be used. For all methods of the invention it is preferred that a DNA isolation method is used that is specifically tested for its applicability for
isolation of DNA from urine. Various commercial DNA isolation kits designed and marketed for use with urine are available. In the present invention a method of the invention use such a commercial kit.
Various methods for differentially detecting methylated DNA are available to the skilled person. Non-limiting examples are:
• Methylation-Specific PCR (MSP) or quantitative Methylation-Specific PCR (qMSP), which is based on a chemical reaction of sodium bisulfite with DNA that converts unmethylated cytosines of CpG dinucleotides to uracil or UpG, followed by traditional PCR. However, methylated cytosines will not be converted in this process, and primers are designed to overlap the CpG site of interest, which allows one to determine methylation status as methylated or unmethylated. As an example the EZ DNA Methylation™ kit (Zymo Research, Orange, CA, USA) can be used to convert isolated DNA.
• Whole genome bisulfite sequencing, also known as BS-Seq, which is a high- throughput genome-wide analysis of DNA methylation. It is based on aforementioned sodium bisulfite conversion of genomic DNA which is then sequenced on a Next-generation sequencing platform. The sequences obtained are then re-aligned to the reference genome to determine methylation states of CpG dinucleotides based on mismatches resulting from the conversion of unmethylated cytosines into uracil.
• The HELP assay, which is based on restriction enzymes’ differential ability to recognize and cleave methylated and unmethylated CpG DNA sites.
• ChlP-on-chip assays, which is based on the ability of commercially prepared antibodies to bind to DNA methylation-associated proteins like MeCP2.
• Restriction landmark genomic scanning, a complicated and now rarely-used assay based upon restriction enzymes' differential recognition of methylated and unmethylated CpG sites; the assay is similar in concept to the HELP assay.
• Methylated DNA immunoprecipitation (MeDIP), analogous to chromatin immunoprecipitation, immunoprecipitation is used to isolate methylated DNA fragments for input into DNA detection methods such as DNA microarrays (MeDIP-chip) or DNA sequencing (MeDIP-seq).
• Pyrosequencing of bisulfite treated DNA. This is sequencing of an amplicon made by a normal forward primer but a biotinylated reverse primer to PCR the gene of choice. The Pyrosequencer then analyses the sample by denaturing the DNA and adding one nucleotide at a time to the mix according to a sequence given by the user. If there is a mismatch, it is recorded and the percentage of DNA for which the mismatch is present is noted. This gives the user a percentage methylation per CpG island.
• Molecular break light assay for DNA adenine methyltransferase activity - an assay that relies on the specificity of the restriction enzyme Dpnl for fully methylated (adenine methylation) GATC sites in an oligonucleotide labeled with a fluorophore and quencher. The adenine methyltransferase methylates the oligonucleotide making it a substrate for Dpnl. Cutting of the oligonucleotide by Dpnl gives rise to a fluorescence increase.
• Methyl Sensitive Southern Blotting is similar to the HELP assay, although uses Southern blotting techniques to probe gene-specific differences in methylation using
restriction digests. This technique is used to evaluate local methylation near the binding site for the probe.
• Methylated DNA sequencing, such a genome wide sequendng of DNA digested by methylation sensitive restriction enzymes.
• DNA methylation analysis using nanotechnology, lab-on-a-chip analysis or CRISPR- Cas based methods.
In one embodiment a method for DNA methylation detection and/or quantification comprises isolating cell free DNA and enriching said nucleic add for fragments of a size of 150 nudeic add bases or less, treating said isolated DNA with bisulfite and performing quantitative methylation-specific PCR (qMSP).
Collection of nucleic acid from a sample of a human subject is done from at least 10 ml, preferably at least 20 ml of urine of said subject. Often more than 10 or 20 ml is used. It is preferred to collect nucleic acid from at least 40 ml of urine. In some embodiments nucleic acid is collected from at least 50, 75 or 100 ml of urine. Urine is typically sampled and provided with ethylenediaminetetraacetic acid (EDTA) as soon as possible. Preferably the collection cup contains EDTA in amounts sufficient to reach a concentration of at least 40 mM EDTA. A kit according to the present invention thus preferably comprises a urine collection cup comprising EDTA is an amount sufficient to allow for a dissolved concentration of EDTA of 40 mM EDTA in a full cup. Fresh collected urine is preferably centrifuged with a g-foree routinely used to pellet cells from the urine. The thus obtained cell free urine is subsequently processed immediately for the collection of “cell free” nucleic acid or stored at 4 °C for later processing. Both addition of EDTA and storage at 4 °C preserves urine DNA for accurate methylation analysis. If storage for more than 3 days is contemplated it is preferred that the above treated urine sample is stored at -20°C or at -80°C.
With “cell free” nucleic acid is meant nudeic add isolated from urine from which cells have been removed prior to collection of the nudeic add. Centrifugation with a g-force routinely used to pellet cells from the urine, as mentioned above, is a suitable method to pre-treat urine for the preparation of cell-free nudeic add. It is certainly not the only method. One may use filtration or nudeic add specific magnetic beads or yet other means, for instance.
Processing of the sample to obtain cell free nucleic acid enriched for fragments of 150 nucleic add bases or less can be done using any means suitable for the purpose. In the present invention the processing is preferably done by centrifugation followed by collection of supernatant and DNA isolation thereof to obtain a nucleic add sample enriched for DNA fragments of 150 nucleic acid bases or less. Preferably the nucleic add sample is enriched for fragments of a length of 150-60 nucleic add bases. Such enriched nucleic add samples are well suited for determining, for instance via amplification, the presence of (methylated) fragments according to the invention. The collection methods may be developed or used that enrich for fragments of other sizes, these are also suited for the present invention as long as they allow significant detection/quantification of relevant methylation. Typically such methods must allow the enrichment of fragments around the 100 bases, preferably of 130-
70 nudeic add bases in length. Enrichment is expressed relative to total cell free nudeic add in the sample. Typically the enrichment is such that the enriched nudeic add contains relatively more nudeic add of a size of 150 nucleic add bases or less than centrifuged urine prior to the enrichment step. Nucleic add that is enriched for smaller fragments as indicated herein above, is also referred to as “enriched nucleic add”. Various methods are available for size fractionating nudeic add. Suitable methods are detailed in the examples. A preferred method comprises centrifugation or the use of commercially available nudeic add fragment size selection kit such as the Select-a-Size DNA Clean & Concentrator MagBead Kit (Zymo Research, Irvine, CA, US).
As demonstrated in the examples, excellent results were obtained with the method according to the invention, comprising a step of centrifugation of urine to obtain urine supernatant, followed by isolation of said DNA fragments from said supernatant to isolate cell free nucleic add enriched in fragments of 150 nucleic add bases or less.
Without wishing to be bound by any theory, it is believed that methylated fragments in a nucleic add sample collected from the supernatant of centrifugated urine may be detected at a higher accuracy compared to detection of methylated fragments in the low-molecular weight fraction isolated directly from unfractioned urine, i.e. without first enriching the urine sample in short DNA fragments. Without being bound by theory, it is believed that with the preferred method according to the invention, superior enrichment in fragments of 150 nucleic add bases or less may be achieved, thereby increasing the accuracy of detection of methylated DNA fragments in the sample. Preferably herein, said methylated fragments comprise methylated nudeic add of the SEPTJN-9 gene or a promoter region thereof and methylated nucleic add of the SDC2 gene or a promoter region thereof.
Nucleic acid detected in the urine is typically DNA or a modification thereof as typically may occurs in the body of a human subject. The DNA is typically genomic DNA or more particular a fragment of genomic DNA.
Determining whether enriched nucleic add comprises methylated nudeic add of the SEPTIN-9 gene or a promoter region thereof and methylated nudeic add of the SDC2 gene or a promoter region thereof, can be done in various ways. Detection is typically a yes or no result, typically above or below the detection limit of the particular method used. Determining the level of methylation is a suitable method for the detection of methylation. Detecting any level of methylation other than zero is than similar to detecting as defined above. Determining the level presupposes the use of a method suitable for quantification of the result. Various methods for the quantification are available and known to the person skilled in the art (see above). Different methods typically have their own associated sensitivity and discrimination level.
In the present invention, the level is preferably determined by comparison to a reference. Typically the reference is a value indicative for the quality and/or quantity of the collected enriched nucleic acid. Often this is nucleic add of a genomic sequence that is known to be present at relatively constant levels in the mentioned enriched nucleic add. In the present
invention it is preferred that this reference is nucleic acid of the ACTB gene. Preferably the enriched nucleic acid is amplified with a primer pair of which the forward and reverse primers hybridize under stringent conditions to respectively the nudeic acid sequence of SEQ ID NO: 7, or a complement thereof, and the nudeic add sequence of SEQ ID NO: 8 or a complement thereof. The amplified fragment is preferably detected using a probe with a sequence of SEQ ID NO: 9 or a complement thereof.
With a complement of a given sequence is meant the reverse complement of the indicated sequence. The reverse complement is the sequence that can hybridize to the indicated sequence.
A method for detecting the presence and/or the level of the indicated nucleic adds as mentioned herein preferably further comprises predicting from the presence and/or the level of said methylated SEPTIN-9 and SDC2 nucleic add whether said subject has colorectal cancer, a colorectal metastasis, a recurrent colorectal cancer or a combination thereof. As such the methods can be used as a diagnostic.
The invention further provides a method of determining whether a human subject has colorectal cancer, a colorectal metastasis, a recurrent colorectal cancer or a combination thereof comprising collecting nucleic add from a sample comprising at least 10 ml, preferably at least 20 ml of urine of said subject, processing the nudeic add to isolate cell free nudeic add enriched for fragments of 150 nudeic add bases or less, and determining whether the enriched nucleic add comprises methylated nudeic acid of the SEPTIN-9 gene or a promoter region thereof and methylated nucleic add of the SDC2 gene or a promoter region thereof and determining that said subject has colorectal cancer, a colorectal metastasis, a recurrent colorectal cancer or a combination thereof based on the presence and/or the level of said methylated nudeic add.
The invention further provides a method of providing a human subject in need thereof with a treatment of colorectal cancer, a colorectal metastasis, a recurrent colorectal cancer or a combination thereof, the method comprising: providing a urine sample of at least 10 ml, preferably at least 20 ml of the subject, isolating cell free nucleic add enriched for DNA fragments of 150 nudeic acid bases or less from said urine; detecting the presence or the level of methylation of the SEPTIN-9 gene or a promoter region thereof and the SDC2 gene or a promoter region thereof in said enriched nucleic add, determining from the presence and or the level of said methylation that the human subject has colorectal cancer, a colorectal metastasis, a recurrent colorectal cancer or a combination thereof, and providing a treatment for said colorectal cancer, a colorectal metastasis, a recurrent colorectal cancer or a combination thereof to the subject in need thereof. The cancer treatment is preferably tumor resection, chemotherapy, radiotherapy, immunotherapy or targeted therapy such as with an antibody such as Bevacuzimab. The cancer treatment can also include a treatment to prevent the development of cancer such as with adenoma. Adenoma and particularly advanced adenoma are often removed surgically via endoscopic intervention.
The terms tumor and cancer are used interchangeably herein. Tumor resection indudes removal of cancer tissue or adenoma by surgery and removal of cancer tissue or adenoma via endoscopic intervention.
Detecting and/or determining the level of certain DNA molecules in a sample can be done using various methods. Often but not necessarily these method involve a nucleic acid amplification step (further referred to as amplification). Such amplification may be the generation of one copy or many copies. PCR and many other methods of amplification typically generated many copies. In one embodiment the detecting and/or determining the level of comprises performing a nucleic add amplification step with said enriched nucleic add. Present day nucleic add sequencing techniques can be as sensitive as the detection of one DNA molecule, so in some embodiments detection and/or determining the level comprises such a highly sensitive sequencing step. Suitable methods involve a single molecule sequence step such as a method referred to as nanopore sequencing.
A method for detecting and/or determining the level of preferably comprises treating said enriched nudeic add with bisulfite prior to performing a nucleic add amplification.
An amplification step as indicated herein preferably comprises nucleic acid amplification with a first and/or second primer that spedfically hybridizes under stringent conditions to the nudeic acid sequence of SEPTIN-9 and a first and/or a second primer that specifically hybridized under stringent conditions to the nudeic acid sequence of SDC2.
Particularly preferred is an amplification step comprising nucleic acid amplification with a first and a second primer pair of which the primers of the first primer pair hybridize under stringent conditions to respectively the nudeic add sequence of SEQ ID NO: 1, or a complement thereof and the nucleic add sequence of SEQ ID NO: 2 or a complement thereof, and the primers of the second primer pair hybridize under stringent conditions to respectively the nudeic add sequence of SEQ ID NO: 4, or a complement thereof, and to the nudeic add sequence of SEQ ID NO: 5 or a complement thereof Nudeic add amplified in this way is preferably probed with a first probe that hybridizes under stringent conditions to the nudeic add sequence of SEQ ID NO: 3 and a second probe that hybridizes under stringent conditions to the nucleic add sequence of SEQ ID NO: 6. The determined level is preferably compared with a reference. The reference is preferably amplified ACTB nucleic add. The amplification of ACTB nudeic add is preferably done with a primer pair of which the forward and reverse primers hybridize under stringent conditions to respectively the nudeic add sequence of SEQ ID NO: 7, or a complement thereof, and the nucleic add sequence of SEQ ID NO: 8 or a complement thereof. The amplified fragment is preferably detected using a probe with a sequence of SEQ ID NO: 9 or a complement thereof
Whether or not DNA methylation is higher than or similar to a reference value can be determined using statistical methods that are appropriate and well-known in the art, generally with a probability value of less than five percent chance of the change being due to random variation. It is well within the ability of a skilled person to determine the amount of increase or similarity that is considered significant. Preferably, “higher than” is
at least 20, at least 40, or at least 50% higher than the reference value. Preferably, similar to” is at most 20% difference, more preferably at most 10% difference between DNA methylation determined and the reference value(s).
The invention further provides a kit of parts comprising means for the detection of DNA methylation of nucleic add of at most 10 genes or promoter regions thereof comprising wherein two of said genes or promoter regions thereof are SEPTIN-9 and SDC2. Preferably, said means comprise a first and/or second primer that specifically hybridizes under stringent conditions to the nucleic add sequence of SEPTIN-9 and a first and/or a second primer that specifically hybridizes under stringent conditions to the nucleic acid sequence of SDC2. It preferably comprises a first and a second primer pair of which the primers of the first primer pair hybridize under stringent conditions to respectively the nucleic add sequence of SEQ ID NO: 1, or a complement thereof and the nucleic acid sequence of SEQ ID NO: 2 or a complement thereof, and the primers of the second primer pair hybridize under stringent conditions to respectively the nucleic add sequence of SEQ ID NO: 4, or a complement thereof and to the nudeic add sequence of SEQ ID NO: 5 or a complement thereof.
The kit of parts preferably further comprises a first probe that specifically hybridizes under stringent conditions to the nudeic add sequence of SEPTIN-9 and a second probe that specifically hybridizes under stringent conditions to the nucleic acid sequence of SDC2.
The kit of parte preferably further comprises a first and a second probe wherein said first probe hybridizes under stringent conditions to the nudeic add sequence of SEQ ID NO: 3 and said second probe hybridizes under stringent conditions to the nucleic add sequence of SEQ ID NO: 6. The kit may further comprise a DNA isolation kit specifically designed to collect nudeic add from urine, as indicated herein above.
Such a kit may comprise one or more of the following components: a container for collecting urine, a container filled with EDTA to achieve an EDTA concentration of at least 40 mM and/or one or more other additives, and test tubes for analysis. Said means for detection of DNA methylation may comprise primers and optionally a probe suitable for MSP or qMSP of the genes disclosed herein or a promoter region thereof, preferably primers as described herein, and/or methylation-sensitive restriction enzymes. Preferably, said means comprise primers suitable for determining DNA methylation of a SEPTIN-9 promoter and SDC2 promoter. Said means for detection of DNA methylation may further comprise means for isolating DNA, preferably genomic DNA and/or bisulfite for converting isolated methylated DNA
In one embodiment the kit of part is for use in a method of diagnosing colorectal cancer, a colorectal metastasis, a recurrent colorectal cancer or a combination thereof in a subject, the method comprising collecting nucleic acid from a sample comprising at least 10 ml, preferably at least 20 ml of urine of said subject, processing the nucleic acid to isolate cell free nucleic acid enriched for fragments of 150 nucleic acid bases or less, and determining
whether the enriched nucleic add comprises methylated nucleic acid of the SEPTIN 9 gene or a promoter region thereof and the SDC2 gene or a promoter region thereof.
For the purpose of clarity and a concise description features are described herein as part of the same or separate embodiments, however, it will be appreciated that the scope of the invention may include embodiments having combinations of aB or some of the features described.
A reference herein to a patent document or other matter which is given as prior art is not to be taken as an admission that that document or matter was known or that the information it contains was part of the common general knowledge at the priority date of any of the claims.
AB patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.
The invention is further explained in the following examples. These examples do not limit, the scope of the invention, but merely serve to clarify the invention.
Further definitions:
As used herein, "to comprise” and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition the verb “to consist” may be replaced by “to consist essentially of’ meaning that a compound or adjunct compound as defined herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention.
The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.
The word “approximately” or “about” when used in association with a numerical value (approximately 10, about 10) preferably means that the value may be the given value of 10 more or less 1% of the value.
As used herein, the terms " treatment," "treat," and "treating” refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease or disorder, or one or more symptoms thereof, as described herein. In some embodiments, treatment may be administered after one or more symptoms have developed. In other embodiments, treatment may be administered in the absence of symptoms. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example to prevent or delay their recurrence.
Optionally, a method of the invention for determining a treatment strategy or for treatment of an individual comprises determining the grade or stage of the cancer. Suitable methods for determining the grade and/or stage of cancer include obtaining and analyzing one or more biopsies and imaging of the cancer, e.g. using CT, MRI, x-rays, PET scan, etc. A physician or other health care professional can readily determine a suitable treatment option, such as surgery, chemotherapy, targeted therapy, radiation therapy, endoscopic interventions and immunotherapy, or a combination of one or more of said treatment options.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Methylation levels (a + c) and detection rates (b + d) in both unfractioned urine and urine supernatant samples. Figure la. and 1c. shows methylation detection of SEPT-9, TMEFF2, SDC2, NDRG4, VIM and ALX4 in unfractioned and supernatant urine samples of CRC patients and controls. Data are shown as the median with interquartile range of 21og converted Ct ratios. In figure lb. + 1d., detection rates of markers in unfractioned urines samples of CRC patients and controls are depicted, meaning the percentage of samples scoring a CtMARKER value below 45.
Figure 2. SEPT-9 methylation levels in urine supernatant per CRC stage. Samples of patient with stage IV CRC have been further stratified in patients with and without the primary tumor still being present. Furthermore, methylation levels in patients with solely intraperitoneal metastases (n=15) and patients diagnoses with stage IV due to liver metastases (n=5) are shown. Data are shown as the median with interquartile range of 21og converted Ct ratios.
Figure 3: The CART decision tree. The boxes depict the decision nodes. Based on SEPT- 9 or SDC2 methylation values, samples are classified as a case (1) or control (0). The numbers below the node represent urine samples that are classified incorrectly (red) or correctly (green) according to the classification of 0 or 1. Node numbers are indicated above by 1 to 7.
Figure 4: The ROC curve for classification of supernatant urine samples using multivariate logistics regression (MLR) model with marker SEPT-9 and interaction of markers SEPT-9 and SDC2. The red diamond indicates the maximal Youden’s index.
Figure 5. The performance of both generated models and their accuracy in detection of CRC in supernatant urine. The figure shows the classification of CRC samples and controls for both MLR and CART models. At the bottom, performance specifics for both models are depicted.
EXAMPLES
MATERIAL & METHODS
Study subjects and sample processing
All patients were older than 18 years, were diagnosed with a pathology proven CRC, and underwent no recent anticancer treatment during the last year. Patients with other malignancies in the previous 3 years were excluded. All participating patients provided urine samples during visits to the outpatient clinic prior to surgery. Samples were collected in 40ml containers containing 40mM Ethylenediaminetetraacetic add (EDTA) and subsequently processed within 6 hours and stored at 4°C. Both addition of EDTA and storage at 4 °C preserves urine DNA for accurate methylation analysis 24. Healthy volunteers, serving as controls, were selected for eligibility through a pre-defined selection process. By taking a questionnaire, it was verified if control subjects were not diagnosed with cancer at any point during their life, and matched the age range of the CRC patient test groups. Urine samples from controls were also collected in containers containing 40mM EDTA and processed upon arrival. For both CRC patients and controls an independent set of consecutively collected urine samples was used for our studies on unfractioned urine and urine supernatant. To obtain the urine supernatant fraction and enrich for low-MW DNA samples were centrifuged at 3000g for 15 minutes. Unfractioned and supernatant urine specimens were frozen at -20 °C until further use.
DNA isolation and bisulfite modification
For DNA isolation from unfractioned urine and urine supernatant the Quick DNA urine kit (Zymo Research, Irvine, CA, US) was used. Isolated DNA was eluted in 50 ul elution buffer. DNA concentrations were measured using the Qubit™ dsDNA HS Assay (Invitrogen, Carlsbad, CA, US). For methylation analysis, up to 400 ng of isolated DNA was treated with bisulfite using the EZ DNA Methylation kit (Zymo Research, Irvine, CA, US). All procedures were performed according to manufacturer's guidelines.
Quantitative methylation specific PCR (qMSP)
Six CRC- associated DNA methylation markers (SEPT-9, TMEFF2, SDC2 and NDRG4, VIM and ALX4) were selected from a systematic literature search based on accuracy 3. Two multiplex quantitative Methylation Specific PCRs (qMSPs), each consisting of 3 targets (SEPT- 9, TMEFF2 & SDC2 and NDRG4, VIM.& ALX4) and reference gene (B-actin: ACTB) were designed based on sequences as described previously 25 -29. By adjusting amplicon sizes to a maximum of 80 bp, detection of CRC-derived low-MW DNA was facilitated. Methylation marker abundance was calculated relative to ACTB levels (Ct- ratio), using the following formula: 2 - (CtMARKER - CtACTB) * 100.
Multiplex amplifications were performed with the primers and probes listed below
+ = Locked nucleic acid (LNA). +MGB = Minor Groove Binding probe. Oligonucleotides were obtained from Eurogentec.
Data analysis
Ct-ratios of methylation targets were compared between groups using the Mann Whitney U test. Results from statistical tests were corrected for multiple testing by the Bonferroni- procedure. Differences in absolute detection rates were compared and tested for statistical significance with the Pearson’s Chi-square test. P values <0.05, adjusted using Bonferroni correction, were considered to be statistically significant.
Analyses of relationships between methylation and clinical parameters were only performed for marker SEPT-9, since all other markers did not have sufficient data points for additional statistical analysis. In the patient group, SEPT-9 was compared to cancer stage by the Kruskal Wallis test. Due a large proportion of stage IV patients in our study having only peritoneal metastases (68%), results of stage IV patients were split up between peritoneal metastasis solely and stage IV including all types of (including hematogenous) metastasis. In this study these were only liver metastases.
To determine the ability of a combination of markers to differentiate between controls and patients, two approaches were explored for determining both the best marker panel and marker thresholds for a maximal test accuracy. In the first method, multivariate logistics regression (MLR) was used to model the probability of a urine sample being from a CRC patient, with all six methylation markers as independent variables. First, a model was fit with the six main effects only, and selected markers by stepwise selection. Then, to investigate whether the in-model effect of an individual marker was affected by other
markers, the two way interaction terms were added that include the selected main effects, again followed by stepwise selection. A leave-one-out cross-validation was then used to evaluate the performance of the model for prediction. Next, the predicted probability from this cross-validation was used for sample classification, according to a maximal Youden’s index, ie. the sum of sensitivity and specificity minus 1. For fitting the MLR model, the R function Generalized Linear Models or glm was used. Apart from the MLR, we applied an algorithm-based method called classification and regression tree (CART) for binary classification of cases and controls on the same set of methylation markers. For this alternative analysis, a decision tree was obtained allowing for classification of urine samples based on marker values. For further details on CART method see 31. For the purpose of prediction, the predicted class was obtained by leave-one-out cross validation. For both building the decision tree as well as performing prediction, the R package Recursive Partitioning or rpart was used.
The performance of both methods was determined from obtained sensitivities and specificities. For logistics regression, the Receiver Operator Characteristic (ROC) curve was plotted together with maximized Youden’s index.
Statistical analyses were performed using SPSS software (SPSS 22.0, IBM, Armonk, NY, USA) and R (Vienna, Austria. UR). Data visualization and construction of graphs was facilitated by GraphPad (Graphpad Prism version 8.2.1, La Jolla, CA USA).
Results
Patient and sample characteristics
In total 47 CRC patients and 20 healthy controls were included in the unfractioned group, and 45 CRC patients and 43 controls in the supernatant group. Clinical characteristics of CRC patients and controls with valid qMSP results are depicted in Table 1.
Table 1. Overview of characteristics of included patients.
The DNA yield of both unfractioned urine and urine supernatant collected from CRC patients and controls were evaluated to assess the utility for methylation analysis. The sample DNA concentrations are shown in Table 2. Concentrations of unfractioned urine samples were approximately three to five times higher as compared to supernatant samples, for patients and controls. Regarding sex, unfractioned urine DNA concentrations of female subjects were two times higher than DNA concentrations measured in male subjects.
Table 2. Median urine DNA concentrations for unfractioned and supernatant samples of 40ml.
DMA methylation detection rates in unfractioned urines samples
DNA methylation of SEPT-9, TMEFF2, SDC2, NDRG4, VIM and ALX4 in unfractioned urine samples of CRC patients (n = 47) and controls (n = 20) was investigated to evaluate their potential for CRC detection. Elevated methylation levels were detected in a subset of CRC patients for SEPT-9, and at low frequencies for VIM and ALX4 (Figure la). Following
Bonferroni correction, none of the markers was found to be significantly different between patients and controls. Likewise, no significant differences were seen for methylation detection rates, defined as any positive signal in qMSP analysis (i.e. Ct value <45)(Figure lb). Methylation marker SEPT-9 was detected in all CRC patients as well as nearly all controls (90%). The remaining markers were detectable in 2-36% of CRC patients and 0- 20% of controls.
DNA methylation detection rates in urine supernatant samples
Next we determined if the supernatant firaction, which is presumed to be enriched for cfDNA 18, would allow for a better discrimination between patients and controls. The same methylation markers were tested on urine supernatants from an independent cohort of CRC patients (n=45) and controls (n=44). As shown in Figure 1c SEPT-9 methylation levels were significantly elevated in CRC patients compared to controls (p<0.0001). No significant differences were found for the remaining five markers. Assessment of the absolute detections rates also demonstrated that SEPT-9 methylation analysis detected significantly more CRC patients than controls (p<0.01)(Figure Id). No differences in detection rates between the two groups were found for the other five markers.
In the group of CRC patients, no difference in SEPT-9 methylation levels was found between the different clinical stages of CRC disease (Figure 2). However, within the stage IV patients, SEPT-9 methylation levels were significantly increased in patients of which the primary tumor was still present during urine collection, compared to stage IV patients having a history of resection of the primary CRC tumor (p<0.01). Patients with solely peritoneal metastases had a trend towards lower levels of urine ctDNA, as compared to patients with liver metastases.
Discriminating potential of combined methylation markers in urine supernatant The diagnostic potential of a combined panel of DNA methylation markers was evaluated to differentiate between healthy controls and CRC patients. Both a multivariate logistics regression (MLR) and classification and regression tree (CART) analysis methods were used to assess accuracy. Furthermore, discrepancies were determined between models with regard to sample classification. To allow for a complete analysis, CRC samples (n=2) and control samples (n=l) that had an invalid ACTB in one of the two multiplexes, were discarded in this process which resulted in a total of 43 CRC and 42 control urine supernatants to be evaluated.
When fitting the MLR model with main effects, only SEPT-9 was significantly associated with the probability of being in the case group (p<0.0001). Therefore, a logistics regression was fitted, with SEPT-9 and the interaction terms with the other five markers. The stepwise selection procedures selected SEPT-9 methylation and the interaction term between SEPT-9 and SDC2 methylation to be strongly associated with the probability of being a CRC patient. This illustrates the behavior of the estimated probability of being a CRC case for various values of SEPT-9 and SDC2. In this model, when methylation levels of SEPT-9 were low, the probability of being a case was small, irrespective of SDC2 levels. In the higher values of SEPT-9 however, we noticed that gradual increases of predicted probabilities of being a case were affected by the values of SDC2. This explains the interactive effect of SEPT-9 and SDC2.
In the second approach, we used a CART model to classify cases and controls based on the values of each markers. The resulting decision tree is depicted in figure 3. As in the logistics regression, SEPT-9 is the most important predictor for the classification, but again SDC2 constitutes an interaction variable. When SEPT-9 methylation was higher or equal to -0.098, subjects were classified into cases (Figure 3, node 3). In node 3, there are 29 correctly classified and 4 misclassified subjects. In the next step, subjects were classified into controls when having SEPT-9 methylation lower than -2.9. In this branch, 27 subjects were correctly classified and 5 subjects were misclassified (node 4). Finally, when the value of SEPT-9 was lower than -2.9, the threshold of SDC2 = -1.8 determined the controls (i.e. SDC2 ≤ -1.8, with 9 correctly classified and 4 misclassified subjects) and cases (i.e. SDC2 > - 1.8, with 5 correctly and 2 misclassified subjects).
Finally, leave-one-out cross validation was utilized to evaluate the prediction performance of MLR and CART. Using the MLR model, we obtained the estimated probability of being a case while the CART decision tree assigns a sample as ease or control. Hence, unlike CART decision tree, logistic regression allowed drawing a receiver operating characteristic (ROC)- curve (Figure 4). A maximized Youden’s index was used to compare the performances of both methods. Figure 5 illustrates the performance of both models. In general, the performance of both MLR with interaction and CART were almost similar. While the MLR provided slightly higher sensitivity compared to CART (70% vs 67%), the latter had a slightly better specificity (88% vs 86%). Furthermore, the two models agreed on the classification of >90% of all samples (Figure 5). Both models were also able to detect CRC independent of cancer stage.
Discussion
The inventors demonstrate for the first time that urine of CRC patients contains elevated levels of the DNA methylation marker SEPT-9, as compared to healthy control patients. SEPT-9 methylation, combined with marker SDC2, offers a potential novel tool for detection and monitoring of CRC. Using short-amplicon methylation specific PCRs, they successfully detected CRC-associated DNA methylation in urine supernatant. Out of six markers tested, SEPT-9 showed best accuracy to serve as a potential urinary biomarker for CRC detection. By combining SEPT-9 and SDC2, up to 70% of CRC cases could be detected at a specificity of 86%.
Despite extensive research, a need still exists for a non-invasive biomarker to detect CRC during clinical management While many studies have been performed on the use of ctDNA in plasma for these purposes, a possible role for urine has not yet been well elucidated. Urine as a biofluid poses several advantages over blood. It does not require trained professionals to acquire, it lends itself for easy repeated sampling and it has been shown that urine poses a very stable medium for DNA 24. This allows for reliable testing of samples collected in an ambulant setting. Additionally, there are no limits to available quantities. For screening programs of other types of cancer, urine is currently evaluated as a non-invasive alternative to physician-involved diagnostics 32-35. The results from the present invention show that urine has the same potential for CRC detection, by showing ctDNA is detectable through means of DNA methylation analysis. It is further shown that methylation markers in the blood may have diagnostic value when tested in the context of
CRC, the same is not true for the same markers in urine. Whether or not a marker teste good for use in the urine must be tested specifically and cannot be inferred from data obtained in blood samples.
In a study by Su et al., the distinction between high and low-molecular weight (HMW and LMW) urine DNA for CRC detection was made, showing the latter provides higher accuracy for detection of CRC-spedfic KRAS DNA mutations 22. Some methylation markers tested in the present study have been described before for CRC detection in urine samples.
Methylation marker VIM was assessed in two separate studies 28,36. In a study that selected low molecular weight DNA from urine samples using carboxylated magnetic beads, 12 of 17 LMW (71%) urine DNA samples of CRC patients were found to be positive for VIM methylation, compared to two out of 20 (10%) control samples 28. Another earlier publication however, showed a poor performance of VIM methylation detection in urine (Le. 8% sensitivity at 100% specificity). This study also assessed the methylation markers WIF- 1 and ALX4 in urine, for which respectively a sensitivity of 27% and 15% at 99% and 100% specificity was found 36. Detection of NDRG4 methylation in urine has been described earlier by Xiao et al. with 55 of 76 (73%) CRC cases testing positive for urine methylation, at a specificity of 85% based on 36 controls 29.
In the art it is not known what the reasons are for the observed difference in penetration of significant methylated nucleic in the urine. It could be that there are differences in methodology or sample population that may explain the discrepancies in CRC detection rates of methylation markers NDRG4 and VIM. In the present invention, centrifugation appeared effective for enrichment of highly fragmented tumor DNA (low-MW), as supernatant samples enabled a more adequate differentiation between disease and healthy controls compared to unfractioned urine that contains both high-MW and low-MW DNA. Interestingly, all mentioned previous studies did not fractionate urine samples prior to DNA isolation and did not use specialized urine DNA kite. Song et at stored urine samples on -70 °C directly following collection, and isolated for low-MW DNA using magnetic beads- based selection and resin-based DNA isolation after defrosting of the urine samples 28. Xiao et ah isolated DNA directly following collection, however no pre-PCR DNA size selection was performed and no information was given on the isolation method 28. Amiot et al. did not provide any details with regard to urine processing and used the same generic DNA isolation kit for urine, plasma and stool 36. Besides limited data on the use of urine for detection of CRC, little evidence is currently available for prognostication and disease monitoring 38
To our knowledge, the present invention is the first describing detection of SEPT-9 methylation in urine. Urine SEPT -9 methylation analysis showed a sensitivity for CRC detection coming dose to those reported for SEPT-9 methylation analysis in plasma samples, which vary from 75 to 81% (at >96% specificity) 38. SEPT-9 methylation analysis for CRC detection in plasma is now available as an FDA-approved commercial test (Epi ProColon 2.0®, Epigenomics AG Coporation, Berlin, Germany). Established CRC plasma methylation markers tested in this study, other than SEPT -9 and SDC2, showed lower detection rates. It is not known what the reason is for this difference in detection rates. Possible explanations indude both biological and technical causes. Urine as a biofluid
might have properties that lead to decreased detection of certain ctDNA fragments. Pores present in the glomerular basal membrane (GBM) may select not only on molecular weight, but also its net negative electric charge could play a role in preventing blood -urine translocation of the negatively charged DNA 19,40. Other thermodynamic properties of DNA that lead to its polymorphic potential, as well as complex formation with for instance proteins, might also influence the probability of glomerular translocation. As these properties are hugely influenced by particular nucleotide sequences of ctDNA, certain methylation markers might have a decreased performance in urine.
No differences were found between urine SEPT -9 methylation levels and clinical stages of CRC of patients. Although the detection of ctDNA appears more likely when sampling occurs during higher clinical stages of neoplastic disease, this was not the case in the present study. For stage IV patients however, we found that the presence of the primary tumor may be an attributive factor for detecting CRC-assoeiated methylation in urine DNA. This probably also relates to the fact that the majority of included stage IV patients without the primary tumor present were suffering from peritoneal metastases. Previous data shows that patients with peritoneal metastases have a smaller tendency to have detectable ctDNA 41. Furthermore, the majority of peritoneal metastases are classified as CMS4 (Consensus Molecular Subtypes). In this tumor subtype, methylation levels are often low 42,48.
Hence, although it may be anticipated that methylation levels rise with tumor stage, present data show that this is not necessarily the case for urinary methylation markers. Although we aimed for completely aged-matched case and control groups and succeeded in the unfractioned urine cohort, there was a small age difference in the urine supernatant cohort (median age 66 vs 60). Furthermore, no patients with CRC precursor lesions or other cancer types were included. Genome wide methylation analysis of CRC urine specimens could possibly discover additional markers with better performance in urinary cfDNA.
Furthermore, in this invention, 40 ml of urine was used for DNA isolation, being a technical limitation of methodology. Other studies studying urine for cancer detection have utilized larger volumes (up to 120 ml) of urine 38 ,44.
In conclusion, the present invention demonstrates the feasibility of urine supernatant for detection of CRC. Through means of DNA methylation analysis of a marker panel consisting of SEPT-9 and SDC2, CRC could be detected with high accuracy.
SEQUENCES (5’-> 3* and + indicates a locked nucleic acid modification that does not change the sequence)
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Claims
1. A method of detecting or determining the level of nudeic acid in urine of a human subject, the method comprising the steps of collecting nucleic add from a sample of at least 10 ml of urine of said subject, processing the sample to isolate cell free nucleic acid enriched for fragments of 150 nudeic add bases or less, and determining whether the enriched nucleic add comprises methylated nudeic add of the SEPTIN-9 gene or a promoter region thereof and methylated nudeic add of the SDC2 gene or a promoter region thereof.
2. The method of claim 1, further comprising determining the level of said methylated SEPTIN-9 and SDC2 nucleic add.
3. The method of claim 1 or claim 2, further comprising prognosticating from the presence and/or the level of said methylated SEPTIN-9 and SDC2 nucleic acid whether said subject has colorectal cancer, a colorectal metastasis, a recurrent colorectal cancer or a combination thereof.
4. A method of providing a human subject in need thereof with a treatment of colorectal cancer, a colorectal metastasis, a recurrent colorectal cancer or a combination thereof, the method comprising: providing a urine sample of at least 10 ml of the subject, isolating cell free nudeic acid enriched for DNA fragments of 150 nucleic add bases or less from said urine; detecting the presence or the level of methylation of the SEPTIN-9 gene or a promoter region thereof and the SDC2 gene or a promoter region thereof in said enriched nucleic add, determining from the presence and or the level of said methylation that the human subject has colorectal cancer, a colorectal metastasis, a recurrent colorectal cancer or a combination thereof and providing a treatment for said colorectal cancer, a colorectal metastasis, a recurrent colorectal cancer or a combination thereof to the subject in need thereof.
5. The method of claim 4, wherein the cancer treatment is tumor resection, radiotherapy, chemotherapy, targeted therapy or immunotherapy, such as administration of Bevacuzimab.
6. A method of determining whether a human subject has colorectal cancer, a colorectal metastasis, a recurrent colorectal cancer or a combination thereof comprising collecting nucleic acid from a sample comprising at least 10 ml of urine of said subject, processing the nucleic acid to isolate cell free nucleic add enriched for fragments of 150 nucleic add bases or less, and determining whether the enriched nucleic acid comprises methylated nucleic add of the SEPTIN-9 gene or a promoter region thereof and methylated nudeic acid of the SDC2 gene or a promoter region thereof, and determining that said subject has colorectal
cancer, a colorectal metastasis, a recurrent colorectal cancer or a combination thereof based on the presence and/or the level of said methylated nucleic acid in said urine sample.
7. The method of claims 1-6, wherein said detecting and/or determining the level of comprises a sequencing step, preferably a single molecule sequence step.
8. The method of claims 1-7, wherein said detecting and/or determining the level of comprises performing a nucleic add amplification step with said enriched nucleic acid.
9. The method of claims 1-8, further comprising treating said enriched nucleic add with bisulfite prior to performing a nucleic add amplification.
10. The method of claims 1-9, wherein said nucleic acid amplification step comprises nucleic acid amplification with a first and a second primer pair of which the primers of the first primer pair hybridize under stringent conditions to respectively the nucleic acid sequence of SEQ ID NO: 1, or a complement thereof, and the nucleic acid sequence of SEQ ID NO: 2 or a complement thereof, and the primers of the second primer pair hybridize under stringent conditions to respectively the nucleic acid sequence of SEQ ID NO: 4, or a complement thereof, and to the nucleic acid sequence of SEQ ID NO: 5 or a complement thereof.
The method of claims 1-10, wherein the cell free nucleic acid is collected from the supernatant of centrifugated urine.
12. The method of claims 1-11, wherein determining the level of methylated nucleic add of the SEPTIN-9 gene or a promoter region thereof and methylated nucleic add of the SDC2 gene or a promoter region thereof comprises comparing detected levels with a reference.
13. The method of daims 8-11, wherein amplified nucleic add is probed with a first probe that hybridizes under stringent conditions to the nudeic add sequence of SEQ ID NO: 3 and a second probe that hybridizes under stringent conditions to the nucleic add sequence of SEQ ID NO: 6.
14. The method of claims 1-13, wherein said urine sample has been treated with a preservative, preferably ethylenediaminetetraacetic add (EDTA).
15. A kit of parts comprising means for the detection of DNA methylation of nucleic add of at most 10 genes or promoter regions thereof comprising wherein two of said genes or promoter regions thereof are SEPTIN-9 and SDC2.
16. The kit of parts of claim 15, comprising a first and a second primer pair of which the primers of the first primer pair hybridize under stringent conditions to respectively the
nucleic add sequence of SEQ ID NO: 1, or a complement thereof and the nudeic add sequence of SEQ ID NO: 2 or a complement thereof^ and the primers of the second primer pair hybridize under stringent conditions to respectively the nucleic add sequence of SEQ ID NO: 4, or a complement thereof, and to the nudeic add sequence of SEQ ID NO: 5 or a complement thereof.
17. The kit of parts of claim 15 or claim 16, comprising a first and a second probe wherein said first probe hybridizes under stringent conditions to the nudeic add sequence of SEQ ID NO: 3 and said second probe hybridizes under stringent conditions to the nudeic add sequence of SEQ ID NO: 6.
18. The kit of claims 15-17, further comprising a DNA isolation kit specifically designed to collect nucleic acid from urine.
19. A kit of parts according to claims 15-18, for use in a method of diagnosing colorectal cancer, colorectal metastasis, recurrent colorectal cancer or a combination thereof in a human subject, the method comprising collecting nucleic add from a sample comprising at least 10 ml of urine of said human subject, processing the nucleic add to isolate cell free nucleic add enriched for fragments of 150 nudeic add bases or less, and determining whether the enriched nucleic add comprises methylated nucleic add of the SEPTIN-9 gene or a promoter region thereof and the SDC2 gene or a promoter region thereof.
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| WO2018087129A1 (en) * | 2016-11-08 | 2018-05-17 | Region Nordjylland, Aalborg University Hospital | Colorectal cancer methylation markers |
| CN109811056A (en) * | 2019-02-28 | 2019-05-28 | 苏州唯善生物科技有限公司 | For colorectal cancer and its primed probe group and kit of precancerous lesion early diagnosis, detection or screening |
| WO2020019269A1 (en) * | 2018-07-26 | 2020-01-30 | 北京艾克伦医疗科技有限公司 | Method and kit for identifying gastric cancer status |
| EP3744860A1 (en) * | 2018-06-06 | 2020-12-02 | Suzhou Versabio Technologies, Inc. | Primer and probe set for diagnosis, detection, or screening of colorectal cancer |
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2020
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| WO2018087129A1 (en) * | 2016-11-08 | 2018-05-17 | Region Nordjylland, Aalborg University Hospital | Colorectal cancer methylation markers |
| EP3744860A1 (en) * | 2018-06-06 | 2020-12-02 | Suzhou Versabio Technologies, Inc. | Primer and probe set for diagnosis, detection, or screening of colorectal cancer |
| WO2020019269A1 (en) * | 2018-07-26 | 2020-01-30 | 北京艾克伦医疗科技有限公司 | Method and kit for identifying gastric cancer status |
| CN109811056A (en) * | 2019-02-28 | 2019-05-28 | 苏州唯善生物科技有限公司 | For colorectal cancer and its primed probe group and kit of precancerous lesion early diagnosis, detection or screening |
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