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  • C12Q2537/00—Reactions characterised by the reaction format or use of a specific feature
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  • C12Q2537/164—Methylation detection other then bisulfite or methylation sensitive restriction endonucleases
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  • C12Q2600/00—Oligonucleotides characterized by their use
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  • G16B30/00—ICT specially adapted for sequence analysis involving nucleotides or amino acids
  • G16B30/20—Sequence assembly

Definitions

• the invention relates to cancer detection and classification and more particularly to the use of methylome analysis for the same.
• circulating cell-free DNA cfDNA
• cfDNA circulating cell-free DNA
• Use of DNA methylation mapping of cfDNA as a biomarker could have a significant impact in the field of liquid biopsy, as it could allow for the identification of the tissue-of-origin[2], allow for cancer type and subtype classification, and stratify cancer patients in a minimally invasive fashion[3].
• using genome-wide DNA methylation mapping of cfDNA could overcome a critical sensitivity problem in detecting circulating tumor DNA (ctDNA) in patients with early-stage cancer with no radiographic evidence of disease.
• ctDNA detection methods are based on sequencing mutations and have limited sensitivity in part due to the limited number of recurrent mutations available to distinguish between tumor and normal circulating cfDNA[4, 5].
• genome-wide DNA methylation mapping leverages large numbers of epigenetic alterations that may be used to distinguish circulating tumor DNA (ctDNA) from normal circulating cell-free DNA (cfDNA).
• ctDNA circulating tumor DNA
• cfDNA normal circulating cell-free DNA
• some tumor types such as ependymomas, can have extensive DNA methylation aberrations without any significant recurrent somatic mutations[6].
• a method of detecting the presence of DNA from cancer cells in a subject comprising: providing a sample of cell-free DNA from a subject; subjecting the sample to library preparation to permit subsequent sequencing of the cell-free methylated DNA; adding a first amount of filler DNA to the sample, wherein at least a portion of the filler DNA is methylated, then optionally denaturing the sample; capturing cell-free methylated DNA using a binder selective for methylated polynucleotides; sequencing the captured cell-free methylated DNA; comparing the sequences of the captured cell-free methylated DNA to control cell-free methylated DNAs sequences from healthy and cancerous individuals; identifying the presence of DNA from cancer cells if there is a statistically significant similarity between one or more sequences of the captured cell-free methylated DNA and cell-free methylated DNAs sequences from cancerous individuals.
• a method of detecting the presence of DNA from cancer cells and identifying a cancer subtype comprising: receiving sequencing data of cell-free methylated DNA from a subject sample; comparing the sequences of the captured cell-free methylated DNA to control cell-free methylated DNAs sequences from healthy and cancerous individuals; identifying the presence of DNA from cancer cells if there is a statistically significant similarity between one or more sequences of the captured cell-free methylated DNA and cell-free methylated DNAs sequences from cancerous individuals; and if DNA from cancer cells is identified, further identifying the cancer cell tissue of origin and cancer subtype based on the comparison.
• a computer-implemented method of detecting the presence of DNA from cancer cells and identifying a cancer subtype comprising: receiving, at least one processor, sequencing data of cell-free methylated DNA from a subject sample; comparing, at the at least one processor, the sequences of the captured cell-free methylated DNA to control cell-free methylated DNAs sequences from healthy and cancerous individuals; identifying, at the at least one processor, the presence of DNA from cancer cells if there is a statistically significant similarity between one or more sequences of the captured cell-free methylated DNA and cell-free methylated DNA sequences from cancerous individuals and if DNA from cancer cells is identified, further identifying the cancer cell tissue of origin and cancer subtype based on the comparison.
• a computer program product for use in conjunction with a general-purpose computer having a processor and a memory connected to the processor, the computer program product comprising a computer readable storage medium having a computer mechanism encoded thereon, wherein the computer program mechanism may be loaded into the memory of the computer and cause the computer to carry out the method described herein.
• a computer readable medium having stored thereon a data structure for storing the computer program product described herein.
• a device for detecting the presence of DNA from cancer cells and identifying a cancer subtype comprising: at least one processor; and electronic memory in communication with the at one processor, the electronic memory storing processor-executable code that, when executed at the at least one processor, causes the at least one processor to: receive sequencing data of cell-free methylated DNA from a subject sample; compare the sequences of the captured cell- free methylated DNA to control cell-free methylated DNAs sequences from healthy and cancerous individuals; identify the presence of DNA from cancer cells if there is a statistically significant similarity between one or more sequences of the captured cell- free methylated DNA and cell-free methylated DNA sequences from cancerous individuals and if DNA from cancer cells is identified, further identify the cancer cell tissue of origin and cancer subtype based on the comparison.
• a method of detecting the presence of DNA from cancer cells and determining the location of the cancer from which the cancer cells arose from two or more possible organs comprising: providing a sample of cell-free DNA from a subject; capturing cell-free methylated DNA from said sample, using a binder selective for methylated polynucleotides; sequencing the captured cell-free methylated DNA; comparing the sequence patterns of the captured cell-free methylated DNA to DNAs sequence patterns of two or more population(s) of control individuals, each of said two or more populations having localized cancer in a different organ; determining as to which organ the cancer cells arose on the basis of a statistically significant similarity between the pattern of methylation of the cell-free DNA and one of said two or more populations.
• Figure 1 shows methylome analysis of cfDNA is a highly sensitive approach to enrich and detect ctDNA in low amounts of input DNA.
• Hierarchical clustering method Ward.
• D Performance metrics for the plasma cfDNA methylation-based multi-cancer classifier. Area under the receiver operator curve (auROC) shown on the y-axis for each cancer type and healthy donors following 50-fold generation of elastic net machine learning classifiers.
• Figure 3 shows validation of the multi-cancer classifier on independent cohorts.
• Lung cancer subtypes show ability to distinguish between patients harboring tumors with distinct histologies that have prognostic and therapeutic implications (adenocarcinoma vs. squamous carcinoma vs. small cell carcinoma).
• D Heatmap showing the top DMRs that allow accurate discrimination of the IDH gene mutational status in glioblastoma multiforme (GBM) patient plasma samples.
• GBM glioblastoma multiforme
• Figure 5 shows a suitable configured computer device, and associated communications networks, devices, software and firmware to provide a platform for enabling one or more embodiments as described herein.
• Figure 6 shows sequencing saturation analysis and quality controls.
• A) The figure shows the results of the saturation analysis from the Bioconductor package MEDIPS analyzing cfMeDIP-seq data from each replicate for each input concentration from the HCT1 16 DNA fragmented to mimic plasma cfDNA.
• the horizontal dotted line indicates a fold-enrichment ratio threshold of 25. Error bars represent ⁇ 1 s.e.m.
• DNA methylation profiles are cell-type specific and are disrupted in cancer.
• cfDNA circulating cell-free DNA
• DMRs Differentially Methylated Regions
• Methylome analysis of cfDNA is highly sensitive and suitable for detecting circulating tumor DNA (ctDNA) in early stage patients.
• a machine-learning derived classifier using cfDNA methylomes was able to correctly classify 196 plasma samples from patients with 5 cancer types and healthy donors based on cross- validation.
• the classifier was able to correctly classify AML, lung cancer, and healthy donors, as well as both early and late stage lung cancer. Therefore, methylome analysis of cfDNA can be used for non-invasive early stage detection of ctDNA and robustly classify cancer types.
• a method of detecting the presence of DNA from cancer cells in a subject comprising: providing a sample of cell-free DNA from a subject; subjecting the sample to library preparation to permit subsequent sequencing of the cell-free methylated DNA; adding a first amount of filler DNA to the sample, wherein at least a portion of the filler DNA is methylated, then optionally denaturing the sample; capturing cell-free methylated DNA using a binder selective for methylated polynucleotides; sequencing the captured cell-free methylated DNA; comparing the sequences of the captured cell-free methylated DNA to control cell-free methylated DNAs sequences from healthy and cancerous individuals; identifying the presence of DNA from cancer cells if there is a statistically significant similarity between one or more sequences of the captured cell-free methylated DNA and cell-free methylated DNAs sequences from cancerous individuals.
• Cancer has been traditionally classified by tissue of origin - for instance, colorectal cancer, breast cancer, lung cancer, etc.
• tissue of origin for instance, colorectal cancer, breast cancer, lung cancer, etc.
• therapeutic decisions often hinge on the precise subtype of cancer, and it may be necessary for clinicians to identify the subtype prior to initiation of therapy.
• cancer subtyping that may influence therapeutic decisions include (but are not limited to) stage (e.g., early stage lung cancer treated with surgery vs late stage lung cancer treated with chemotherapy), histology (e.g., small cell carcinoma vs adenocarcinoma vs squamous cell carcinoma in lung cancer), gene expression pattern or transcription factor activity (e.g., ER status in breast cancer), copy number aberrations (e.g., HER2 status in breast cancer), specific rearrangements (e.g., FLT3 in AML), specific gene point mutational status (e.g., IDH gene point mutations), and DNA methylation patterns (e.g., MGMT gene promoter methylation in brain cancer).
• stage e.g., early stage lung cancer treated with surgery vs late stage lung cancer treated with chemotherapy
• histology e.g., small cell carcinoma vs adenocarcinoma vs squamous cell carcinoma in lung cancer
• gene expression pattern or transcription factor activity e
• the methods described herein are applicable to a wide variety of cancers, including but not limited to adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain/ens tumors, breast cancer, castleman disease, cervical cancer, colon/rectum cancer, endometrial cancer, esophagus cancer, ewing family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumor (gist), gestational trophoblastic disease, hodgkin disease, kaposi sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, leukemia (acute lymphocytic, acute myeloid, chronic lymphocytic, chronic myeloid, chronic myelomonocytic), liver cancer, lung cancer (non-small cell, small cell, lung carcinoid tumor), lymphoma, lymphoma of the skin, malignant mesothelioma, multiple myeloma, myelody
• NGS next-generation sequencing
• PCR polymerase chain reaction
• Sanger sequencing also available are next-generation sequencing (NGS) techniques, also known as high-throughput sequencing, which includes various sequencing technologies including: lllumina (Solexa) sequencing, Roche 454 sequencing, Ion torrent: Proton / PGM sequencing, SOLiD sequencing.
• lllumina Solexa
• Roche 454 sequencing Ion torrent: Proton / PGM sequencing
• SOLiD sequencing SOLiD sequencing.
• NGS allow for the sequencing of DNA and RNA much more quickly and cheaply than the previously used Sanger sequencing.
• said sequencing is optimized for short read sequencing.
• subject refers to any member of the animal kingdom, preferably a human being and most preferably a human being that has, has had, or is suspected of having prostate cancer.
• Cell-free methylated DNA is DNA that is circulating freely in the blood stream, and are methylated at various known regions of the DNA. Samples, for example, plasma samples can be taken to analyze cell-free methylated DNA. Accordingly, in some embodiments, the sample is the subject's blood or plasma.
• library preparation includes list end-repair, A-tailing, adapter ligation, or any other preparation performed on the cell free DNA to permit subsequent sequencing of DNA.
• fill DNA can be noncoding DNA or it can consist of amplicons.
• DNA samples may be denatured, for example, using sufficient heat.
• the comparison step is based on fit using a statistical classifier.
• Statistical classifiers using DNA methylation data can be used for assigning a sample to a particular disease state, such as cancer type or subtype.
• a classifier would consist of one or more DNA methylation variables (i.e., features) within a statistical model, and the output of the statistical model would have one or more threshold values to distinguish between distinct disease states.
• the particular feature(s) and threshold value(s) that are used in the statistical classifier can be derived from prior knowledge of the cancer types or subtypes, from prior knowledge of the features that are likely to be most informative, from machine learning, or from a combination of two or more of these approaches.
• the classifier is machine learning-derived.
• the classifier is an elastic net classifier, lasso, support vector machine, random forest, or neural network.
• the genomic space that is analyzed can be genome-wide, or preferably restricted to regulatory regions (i.e., FANTOM5 enhancers, CpG Islands, CpG shores and CpG Shelves).
• the percentage of spike-in methylated DNA recovered is included as a covariate to control for pulldown efficiency variation.
• the classifier would preferably consist of differentially methylated regions from pairwise comparisons of each type (or subtype) of interest.
• the control cell-free methylated DNAs sequences from healthy and cancerous individuals are comprised in a database of Differentially Methylated Regions (DMRs) between healthy and cancerous individuals.
• DMRs Differentially Methylated Regions
• control cell-free methylated DNA sequences from healthy and cancerous individuals are limited to those control cell-free methylated DNA sequences which are differentially methylated as between healthy and cancerous individuals in DNA derived from cell-free DNA from bodily fluids, such as from blood serum, cerebral spinal fluid, urine stool, sputum, pleural fluid, ascites, tears, sweat, pap smear fluid, endoscopy brushings fluid, ..etc., preferably from blood plasma.
• the sample has less than 100 ng, 75 ng, or 50 ng of cell-free DNA.
• the first amount of filler DNA comprises about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% methylated filler DNA with remainder being unmethylated filler DNA, and preferably between 5% and 50%, between 10%-40%, or between 15%-30% methylated filler DNA.
• the first amount of filler DNA is from 20 ng to 100 ng, preferably 30 ng to 100 ng, more preferably 50 ng to 100 ng.
• the cell-free DNA from the sample and the first amount of filler DNA together comprises at least 50 ng of total DNA, preferably at least 100 ng of total DNA.
• he filler DNA is 50 bp to 800 bp long, preferably 100 bp to 600 bp long, and more preferably 200 bp to 600 bp long.
• the filler DNA is double stranded.
• the filler DNA is double stranded.
• the filler DNA can be junk DNA.
• the filler DNA may also be endogenous or exogenous DNA.
• the filler DNA is non-human DNA, and in preferred embodiments, ⁇ DNA.
• ⁇ DNA refers to Enterobacteria phage ⁇ DNA.
• the filler DNA has no alignment to human DNA.
• the binder is a protein comprising a Methyl-CpG-binding domain.
• MBD2 protein is a protein comprising a Methyl-CpG-binding domain.
• MBD2 protein is a protein comprising a Methyl-CpG-binding domain.
• MBD2 protein is a protein comprising a Methyl-CpG-binding domain.
• MBD2 protein refers to certain domains of proteins and enzymes that is approximately 70 residues long and binds to DNA that contains one or more symmetrically methylated CpGs.
• MBD of MeCP2, MBD1 , MBD2, MBD4 and BAZ2 mediates binding to DNA, and in cases of MeCP2, MBD1 and MBD2, preferentially to methylated CpG.
• Human proteins MECP2, MBD1 , MBD2, MBD3, and MBD4 comprise a family of nuclear proteins related by the presence in each of a methyl-CpG-binding domain (MBD). Each of these proteins, with the exception of MBD3, is capable of binding specifically to methylated DNA.
• the binder is an antibody and capturing cell-free methylated DNA comprises immunoprecipitating the cell-free methylated DNA using the antibody.
• immunoprecipitation refers a technique of precipitating an antigen (such as polypeptides and nucleotides) out of solution using an antibody that specifically binds to that particular antigen. This process can be used to isolate and concentrate a particular protein or DNA from a sample and requires that the antibody be coupled to a solid substrate at some point in the procedure.
• the solid substrate includes for examples beads, such as magnetic beads. Other types of beads and solid substrates are known in the art.
• One exemplary antibody is 5-MeC antibody.
• the method described herein further comprises the step of adding a second amount of control DNA to the sample.
• the method further comprises the step of adding a second amount of control DNA to the sample for confirming the immunoprecipitation reaction.
• control may comprise both positive and negative control, or at least a positive control.
• the method further comprises the step of adding a second amount of control DNA to the sample for confirming the capture of cell-free methylated DNA.
• identifying the presence of DNA from cancer cells further includes identifying the cancer cell tissue of origin.
• tumor tissue sampling may be challenging or carry significant risks, in which case diagnosing and/or subtyping the cancer without the need for tumor tissue sampling may be desired.
• lung tumor tissue sampling may require invasive procedures such as mediastinoscopy, thoracotomy, or percutaneous needle biopsy; these procedures may result in a need for hospitalization, chest tube, mechanical ventilation, antibiotics, or other medical interventions. Some individuals may not undergo the invasive procedures needed for tumor tissue sampling either because of medical comorbidities or due to preference.
• identifying the cancer cell tissue of origin further includes identifying a cancer subtype.
• the cancer subtype differentiates the cancer based on stage (e.g., early stage lung cancer treated with surgery vs late stage lung cancer treated with chemotherapy), histology (e.g., small cell carcinoma vs adenocarcinoma vs squamous cell carcinoma in lung cancer), gene expression pattern or transcription factor activity (e.g., ER status in breast cancer), copy number aberrations (e.g., HER2 status in breast cancer), specific rearrangements (e.g., FLT3 in AML), specific gene point mutational status (e.g., IDH gene point mutations), and DNA methylation patterns (e.g., MGMT gene promoter methylation in brain cancer).
• stage e.g., early stage lung cancer treated with surgery vs late stage lung cancer treated with chemotherapy
• histology e.g., small cell carcinoma vs adenocarcinoma vs squamous cell carcinoma in lung cancer
• gene expression pattern or transcription factor activity e.g., ER
• comparison in step (f) is carried out genome-wide. In other embodiments, the comparison in step (f) is restricted from genome-wide to specific regulatory regions, such as, but not limited to, FANTOM5 enhancers, CpG Islands, CpG shores, CpG Shelves, or any combination of the foregoing.
• certain steps are carried out by a computer processor.
• a method of detecting the presence of DNA from cancer cells and identifying a cancer subtype comprising: receiving sequencing data of cell-free methylated DNA from a subject sample; comparing the sequences of the captured cell-free methylated DNA to control cell-free methylated DNAs sequences from healthy and cancerous individuals; identifying the presence of DNA from cancer cells if there is a statistically significant similarity between one or more sequences of the captured cell-free methylated DNA and cell-free methylated DNAs sequences from cancerous individuals; and if DNA from cancer cells is identified, further identifying the cancer cell tissue of origin and cancer subtype based on the comparison step.
• a method of detecting the presence of DNA from cancer cells and determining the location of the cancer from which the cancer cells arose from two or more possible organs comprising: providing a sample of cell-free DNA from a subject; capturing cell-free methylated DNA from said sample, using a binder selective for methylated polynucleotides; sequencing the captured cell-free methylated DNA; comparing the sequence patterns of the captured cell-free methylated DNA to DNAs sequence patterns of two or more population(s) of control individuals, each of said two or more populations having localized cancer in a different organ; determining as to which organ the cancer cells arose on the basis of a statistically significant similarity between the pattern of methylation of the cell-free DNA and one of said two or more populations.
• FIG. 5 shows a generic computer device 100 that may include a central processing unit (“CPU") 102 connected to a storage unit 104 and to a random access memory 106.
• the CPU 102 may process an operating system 101 , application program 103, and data 123.
• the operating system 101 , application program 103, and data 123 may be stored in storage unit 104 and loaded into memory 106, as may be required.
• Computer device 100 may further include a graphics processing unit (GPU) 122 which is operatively connected to CPU 102 and to memory 06 to offload intensive image processing calculations from CPU 102 and run these calculations in parallel with CPU 102.
• An operator 107 may interact with the computer device 100 using a video display 108 connected by a video interface 105, and various input/output devices such as a keyboard 1 5, mouse 112, and disk drive or solid state drive 114 connected by an I/O interface 109.
• the mouse 112 may be configured to control movement of a cursor in the video display 108, and to operate various graphical user interface (GUI) controls appearing in the video display 108 with a mouse button.
• GUI graphical user interface
• the disk drive or solid state drive 1 4 may be configured to accept computer readable media 1 16.
• the computer device 100 may form part of a network via a network interface 1 11 , allowing the computer device 100 to communicate with other suitably configured data processing systems (not shown).
• One or more different types of sensors 135 may be used to
• the present system and method may be practiced on virtually any manner of computer device including a desktop computer, laptop computer, tablet computer or wireless handheld.
• the present system and method may also be implemented as a computer- readable/useable medium that includes computer program code to enable one or more computer devices to implement each of the various process steps in a method in accordance with the present invention.
• the computer devices are networked to distribute the various steps of the operation.
• the terms computer-readable medium or computer useable medium comprises one or more of any type of physical embodiment of the program code.
• the computer-readable/useable medium can comprise program code embodied on one or more portable storage articles of manufacture (e.g.
• a computer-implemented method of detecting the presence of DNA from cancer cells and identifying a cancer subtype comprising: receiving, at least one processor, sequencing data of cell-free methylated DNA from a subject sample; comparing, at the at least one processor, the sequences of the captured cell-free methylated DNA to control cell-free methylated DNAs sequences from healthy and cancerous individuals; identifying, at the at least one processor, the presence of DNA from cancer cells if there is a statistically significant similarity between one or more sequences of the captured cell-free methylated DNA and cell-free methylated DNAs sequences from cancerous individuals and if DNA from cancer cells is identified, further identifying the cancer cell tissue of origin and cancer subtype based on the comparison step;
• a computer program product for use in conjunction with a general-purpose computer having a processor and a memory connected to the processor, the computer program product comprising a computer readable storage medium having a computer mechanism encoded thereon, wherein the computer program mechanism may be loaded into the memory of the computer and cause the computer to carry out the method described herein.
• a computer readable medium having stored thereon a data structure for storing the computer program product described herein.
• a device for detecting the presence of DNA from cancer cells and identifying a cancer subtype comprising: at least one processor; and electronic memory in communication with the at one processor, the electronic memory storing processor-executable code that, when executed at the at least one processor, causes the at least one processor to: receive sequencing data of cell-free methylated DNA from a subject sample; compare the sequences of the captured cell- free methylated DNA to control cell-free methylated DNAs sequences from healthy and cancerous individuals; identify the presence of DNA from cancer cells if there is a statistically significant similarity between one or more sequences of the captured cell- free methylated DNA and cell-free methylated DNAs sequences from cancerous individuals and if DNA from cancer cells from is identified, further identify the cancer cell tissue of origin and cancer subtype based on the comparison step.
• processor may be any type of processor, such as, for example, any type of general-purpose microprocessor or microcontroller (e.g., an IntelTM x86, PowerPCTM, ARMTM processor, or the like), a digital signal processing (DSP) processor, an integrated circuit, a field programmable gate array (FPGA), or any combination thereof.
• general-purpose microprocessor or microcontroller e.g., an IntelTM x86, PowerPCTM, ARMTM processor, or the like
• DSP digital signal processing
• FPGA field programmable gate array
• memory may include a suitable combination of any type of computer memory that is located either internally or externally such as, for example, random- access memory (RAM), read-only memory (ROM), compact disc read-only memory (CDROM), electro-optical memory, magneto-optical memory, erasable programmable read-only memory (EPROM), and electrically-erasable programmable read-only memory (EEPROM), or the like.
• RAM random- access memory
• ROM read-only memory
• CDROM compact disc read-only memory
• electro-optical memory magneto-optical memory
• EPROM erasable programmable read-only memory
• EEPROM electrically-erasable programmable read-only memory
• computer readable storage medium (also referred to as a machine- readable medium, a processor-readable medium, or a computer usable medium having a computer-readable program code embodied therein) is a medium capable of storing data in a format readable by a computer or machine.
• the machine-readable medium can be any suitable tangible, non-transitory medium, including magnetic, optical, or electrical storage medium including a diskette, compact disk read only memory (CD-ROM), memory device (volatile or non-volatile), or similar storage mechanism.
• the computer readable storage medium can contain various sets of instructions, code sequences, configuration information, or other data, which, when executed, cause a processor to perform steps in a method according to an embodiment of the disclosure.
• data structure a particular way of organizing data in a computer so that it can be used efficiently.
• Data structures can implement one or more particular abstract data types (ADT), which specify the operations that can be performed on a data structure and the computational complexity of those operations.
• ADT abstract data types
• a data structure is a concrete implementation of the specification provided by an ADT.
• CRC, Breast cancer, and GBM samples were obtained from the University Health Network BioBank; AML samples were obtained from the University Health Network Leukemia BioBank; Lastly, healthy controls were recruited through the Family Medicine Centre at Mount Sinai Hospital (MSH) in Toronto, Canada. All samples collected with patient consent, were obtained with institutional approval from the Research Ethics Board, from University Health Network and Mount Sinai Hospital in Toronto, Canada.
• Specimen Processing - cfDNA EDTA and ACD plasma samples were obtained from the BioBanks and from the Family Medicine Centre at Mount Sinai Hospital (MSH) in Toronto, Canada. All samples were either stored at -80 ° C or in vapour phase liquid nitrogen until use.
• Cell- free DNA was extracted from 0.5-3.5 ml of plasma using the QIAamp Circulating Nucleic Acid Kit (Qiagen). The extracted DNA was quantified through Qubit prior to use.
• cfMeDIP-seq protocol A schematic representation of the cfMeDIP-seq protocol is shown in WO2017/1902 5.
• the DNA samples were subjected to library preparation using the Kapa Hyper Prep Kit (Kapa Biosystems). The manufacturer protocol was followed with some modifications. Briefly, the DNA of interest was added to 0.2 ml_ PCR tube and subjected to end-repair and A-Tailing. Adapter ligation was followed using NEBNext adapter (from the NEBNext Multiplex Oligos for lllumina kit, New England Biolabs) at a final concentration of 0.181 ⁇ , incubated at 20 ° C for 20 mins and purified with AMPure XP beads. The eluted library was digested using the USER enzyme (New England Biolabs Canada) followed by purification with Qiagen MinElute PCR Purification Kit prior to MeDIP.
• the prepared libraries were combined with the pooled methylated/unmethylated ⁇ PCR product to a final DNA amount of 100 ng and subjected to MeDIP using the protocol from Taiwo et al. 2012[7] with some modifications. Briefly, for MeDIP, the Diagenode MagMeDIP kit (Cat# C02010021 ) was used following the manufacturer's protocol with some modifications. After the addition of 0.3 ng of the control methylated and 0.3ng of the control unmethylated A.
• thaliana DNA to complete the total amount of DNA [cfDNA + Filler + Controls] to 100ng
• the samples were heated to 95 ° C for 10 mins, then immediately placed into an ice water bath for 10 mins.
• Each sample was partitioned into two 0.2 mL PCR tubes: one for the 10% input control and the other one for the sample to be subjected to immunoprecipitation.
• the included 5-mC monoclonal antibody 33D3 (Cat#C15200081 ) from the MagMeDIP kit was diluted 1 :15 prior to generating the diluted antibody mix and added to the sample.
• Washed magnetic beads (following manufacturer instructions) were also added prior to incubation at 4 ° C for 17 hours.
• the samples were purified using the Diagenode iPure Kit and eluted in 50 ⁇ of Buffer C.
• the success of the reaction (QC1 ) was validated through qPCR to detect the presence of the spiked-in A. thaliana DNA, ensuring a % recovery of unmethylated spiked-in DNA ⁇ 1 % and the % specificity of the reaction >99% (as calculated by 1 - [recovery of spiked-in unmethylated control DNA over recovery of spiked-in methylated control DNA]), prior to proceeding to the next step.
• the optimal number of cycles to amplify each library was determined through the use of qPCR, after which the samples were amplified using the KAPA HiFi Hotstart Mastermix and the NEBNext multiplex oligos added to a final concentration of 0.3 ⁇ .
• the PCR settings used to amplify the libraries were as follows: activation at 95 ° C for 3 min, followed by predetermined cycles of 98 ° C for 20 sec, 65 ° C for 15 sec and 72 ° C for 30 sec and a final extension of 72 ° C for 1 min.
• the amplified libraries were purified using MinElute PCR purification column and then gel size selected with 3% Nusieve GTG agarose gel to remove any adapter dimers.
• DNA sequencing libraries were constructed from 83 ng of fragmented DNA using the KAPA Hyper Prep Kit (Kapa Biosystems, Wilmington, MA) utilizing NEXTflex-96 DNA Barcode adapters (Bio Scientific, Austin, TX) adapters.
• KAPA Hyper Prep Kit Kapa Biosystems, Wilmington, MA
• NEXTflex-96 DNA Barcode adapters Bio Scientific, Austin, TX
• the barcoded libraries were pooled and then applied the custom hybrid capture library following manufacturer's instructions (IDT xGEN Lockdown protocol version 2.1 ). These fragments were sequenced to >10,000X read coverage using an lllumina HiSeq 2000 instrument. Resulting reads were aligned using bwa-mem and mutation
• CV 10-Fold Cross-Validation
• pan-cancer data from The Cancer Genome Atlas shows large numbers of DMRs between tumor and normal tissues across virtually all tumor types[1 1 ]. Therefore, these findings highlighted that an assay that successfully recovered cancer-specific DNA methylation alterations from ctDNA could serve as a very sensitive tool to detect, classify, and monitor malignant disease with low sequencing-associated costs.
• cfMeDIP-seq cell-free Methylated DNA Immunoprecipitation and high-throughput sequencing
• the cfMeDIP-seq method described here was developed through the modification of an existing low input MeDIP-seq protocol[7] that in our experience is very robust down to 100 ng of input DNA. However, the majority of plasma samples yield much less than 100 ng of DNA.
• exogenous ⁇ DNA (filler DNA) to the adapter-ligated cfDNA library in order to artificially inflate the amount of starting DNA to 100 ng.
• the filler DNA consisted of amplicons similar in size to an adapter-ligated cfDNA library and was composed of unmethylated and in vitro methylated DNA at different CpG densities. The addition of this filler DNA also serves a practical use, as different patients will yield different amounts of cfDNA, allowing for the normalization of input DNA amount to 100 ng. This ensures that the downstream protocol remains exactly the same for all samples regardless of the amount of available cfDNA.
• Cancer DNA is frequently hypermethylated at CpG-rich regions[17]. Since cfMeDIP- seq specifically targets methylated CpG-rich sequences, we hypothesized that ctDNA would be preferentially enriched during the immunoprecipitation procedure. To test this, we generated patient-derived xenografts (PDXs) from two colorectal cancer patients and collected the mouse plasma. Tumor-derived human cfDNA was present at less than 1 % frequency within the total cfDNA pool in the input samples and at 2- fold greater abundance following immunoprecipitation (Figure 1G; Supplementary Table 3). These results suggest that through biased sequencing of ctDNA, the cfMeDIP procedure could further increase ctDNA detection sensitivity.
• PDXs patient-derived xenografts
• Circulating plasma cfDNA methylation profile can distinguish between multiple cancer types and healthy donors
• DNA methylation patterns are tissue-specific, and have been used to stratify cancer patients into clinically relevant disease subgroups in glioblastoma[18], ependymomas[6], colorectal[19], and breast[20, 21], among many other cancer types.
• cfDNA associated profiles could be used to identify tissues-of-origin for multiple tumor types.
• Performance was then evaluated using AUROC (area under the receiver operating characteristic curve) derived from test set samples (held-out during the DMR selection and the subsequent GLMnet training/tuning steps). This process was repeated with 50 different splits of the discovery cohort into training and test sets to mitigate the influence of training-set biases. This culminated in a collection of 50 models for each one-vs other-classes comparison (480 models in total). Hereby, we refer to this collection of models as E50.
• cfDNA methylome profiling with cfMeDIP-seq The ability of cfDNA methylation patterns to accurately represent tissue-of-origin also overcomes limitations of mutation-based assays, wherein specificity for tissues-of- origin may be low due to the recurrent nature of many potential driver mutations across cancers in different tissues[23]. Mutation based assays may also be rendered insensitive by the clonal structure of tumors, where subclonal drivers may be harder to detect by virtue of lower abundance in ctDNA[24]. Mutation based ctDNA approaches are also vulnerable to potential confounding by driver mutations in benign tissues, which have been observed[25], and documented to display evidence of positive selection[26].
• cfMeDIP-seq as an efficient and cost-effective tool with the potential to influence management of cancer and early detection.
• the accuracy and versatility of cfMeDIP-seq may be useful to inform therapeutic decisions in settings where resistance is correlated to epigenetic alterations, such as sensitivity to androgen receptor inhibition in prostate cancer[27].
• the potential opportunities for early diagnosis and screening may be particularly evident in lung cancer, a disease in which screening has already shown clinical utility but for which existing screening tests (i.e., low dose CT scanning) has significant limitations such as ionizing radiation exposure and high false positive rate.
• Table 1 Number of reads and mapping efficiency of sequenced MeDIP-seq (100 ng Rep 1 and Rep 2) and cfMeDIP-seq (10 ng, 5 ng and 1 ng, Rep1 and Rep 2) libraries prepared using various starting inputs of HCT1 16 cell line DNA sheared to mimic cfDNA, to human (Hg19) genome and ⁇ genome. Two biological replicates were use for starting input DNA. For starting inputs less than 100 ng, the samples were topped up with exogenous A DNA to artificially increase the starling amount to 100 ng prior to Me DIP.
• Table 2A Mean coverage of ultra-deep targeted variant sequencing using dilution series of CRC cell line HCT116 DNA into MM cell line MM1.S DNA
• Table 3 Number of reads and mapping efficiency of cfMeDIP-seq libraries of PDX and Input Control samples after aligning to human (Hg19) genome
• KRAS gene mutations are more common in colorectal villous adenomas and in situ carcinomas than in carcinomas. Int J Mol Epidemiol Genet, 2013. 4(1 ): p. 1 -10.

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