DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Track One Status
The request for track 1 status filed February 27, 2026 is granted. Applicant amendment filed 6/26/2026 is acknowledged. Claims 1, 2 and 5-9 have been amended. Claims 3, 4 and 10-15 have been canceled. Claims 16-39 have been added. Claims 1, 2, 5-9 and 16-39 are under examination.
Election/Restrictions
Applicant’s election without traverse of Group I, claims 1, 2, 4-9 and 16-39 in the reply filed on 6/26/2026 is acknowledged.
Priority
This application is a CON of PCT/YS2025/051855 filed 10/21/2025 which claims benefit of 63/713178 filed 10/20/2024 and clams benefit of 63/711,035 filed 10/23/2024.
Information Disclosure Statement
The information disclosure statements (IDS) submitted on 4/3/2026 and 5/5/2026 are acknowledged. The submission is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Drawings
The drawing submitted on 2/27/2026 is acknowledged. The submissions are in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Specification
The disclosure is objected to because of the following informalities:
(a) The disclosure is objected at paragraph [00112] to because it contains an embedded hyperlink and/or other form of browser-executable code. Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01. Appropriate correction is required.
Claim Rejections - 35 USC § 112(a)
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1-2, 5-9, 16-39 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
The claims are draw to a method of processing cell-free deoxyribonucleic acids (cfDNA) from a subject having or suspected of having a cancer, comprising:(a) obtaining cfDNA fragment sequences from the subject, (b) obtaining sequencing reads from said cfDNA fragment sequences that identify: (i) said cfDNA fragment sequences; (ii) 5-methylcytosine (5mC) in said cfDNA fragment sequences; and (iii) 5-hydroxymethylcytosine (5hmC) in said cfDNA fragment sequences; and (c) applying a trained classifier to said sequencing reads thereby determining a cancer status, wherein said trained classifier has been trained on location or abundance information for 5mC and 5hmC in cfDNA fragment sequences indicative of cancer status.
The claims encompass determining any cancer status based on any abundance or any location of 5mC or 5hmC bases in cfDNA fragments in a subject having a cancer type or suspected of having cancer. The claims also encompass determining cancer status based on applying trained classifier, wherein said trained classifiers have been trained on any location or any abundance information for 5mC and 5 hmC in cfDNA fragment sequence
The specification has not provided guidance for the breadth of the claims fully in scope.
The specification teaches in the figure 1 cytosine methylation in mammals and in figure 2, dynamics of 5mC and 5hmC in colorectal cancer (CRC) samples wherein Volcano plot were obtained for differentially methylated regions (DMRs) between stage IV CRC tissues and adjacent matched normal using data from The Cancer Genome Atlas (TCGA). The specification teaches at Figure 3, a depiction of performance metrics for models using cytosine methylation for predicting colorectal cancer. At figure 4, the specification depicts histogram showing the size of hypo- and hyper-methylated DMRs from TCGA tissue data. Figure 5 depicts scatterplots showing mean methylation differences in stage IV CRC tissue (versus adjacent normal tissue) and mean methylation differences in stage 1 (top) and IV (bottom) cfDNA versus healthy control cfDNA. In the Figure 7, the specification teaches depiction of numbers of feature selected by the LASSO classifier when using both 5mC and 5hmC. Figures 8 depicts a method for differentiating and identifying cytosine, mC and hmC in a DNA sequence and glycosylation of 5hmC, deamination of cytosine and obtaining sequence reads. The specification teaches in Figure 9 a general schematic of a computer system. In the example 1, the specification provides a general synopsis of method steps for detection of disease status in a subject based on the claimed method steps. In the example 2, the specification teaches 5-methylcytosine and 5-hydroxymethylcytosine as synergistic biomarkers for early detection of colorectal cancer and provides in Table 2, the specification teaches sensitivity and specificity for CRC state 1 detection as analyzed in example 2. The specification also discusses trained classifier which may embodies training machine learning modules which are discussed in a generalized manner (see paragraphs [0063] – [0082].
While the specification provides in the Table A some sequence of genes and components described therein, the specification does not provide guidance of target regions to determine methylation profile of 5mC and 5hmC for any and every possible cancer embodied by the claims or even the genes sequences recited therein in the Table A. The specification does not provide critical guidance to determine target genomic regions comprised in all possible cancer types that are differently methylated or means of determining with specificity sufficient differently methylated regions associated with any possible sample. The specification does not disclose what sequence regions are relevant to identifying any and every possible cancer type encompassed by the claims. Further the specification does not disclose in what context these sequences are considered hypermethylated versus hypomethylated or what positions are or are noted methylated in order to detect the status of any possible cancer. Further the specification provides general teaching concerning trained classifier as indicated above for determining a cancer status but does not provide any specific evidence concerning data quality and noise, imbalanced methylation site distribution or variation in methylation changes in any possible sample, sequence context effect and data scarcity for possible rare cancers that are encompassed by the claims as probably written (see Yin et al (Bioinformatics, 2024, 40(2), btae046, pages 1-12 which discuss noise in sequencing data and imbalanced methylation site distribution).
The specification lacks written description commensurate in scope with the claims as broadly written. In analysis of the claims for compliance of written description requirement of 35 USC 112, first paragraph, the written description guidelines note regarding genus/species situations that “Satisfactory disclosure of a “representative number” depends on whether one of skill int heart would recognize that the applicant was in possession of the necessary common attributes or features of the elements possessed by the members of the genus in view of the species disclosed therein” (see “Written Description r” Requirements, Federal Register, vol. 66, no, 4, pages 1099-1111, Friday January 5, 2001.).
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1-2, 5-9 and 16-39 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
(a) Claim 1-2, 5-9, 16-39 are indefinite in the claim 1 and claim 36 at the recitation of “wherein said trained classifier has been trained on location information or abundance information for 5mC or 5hmC in cfDNA fragments indicative of cancer (claim 1) or stage I cancer (claim 36)” because it is unclear what steps are performed such that the trained classifier is capable of indicating any status of cancer. A clear interpretation cannot be ascertained.
(b) Claims 5-9 and 16-29 are indefinite and confusing in the claim 5 and subsequent claims dependent thereto at the recitation of “further comprising attaching a hairpin to strands of said cfDNA fragment sequence…” because it is unclear at which point in the order of steps of (a) –(c) the attaching a hairpin strand and additional limitations recited in the dependent claims are to occur. Likewise, multiple chemical and enzymatic reactions are recited in the dependent claims which further raises ambiguity as the metes of bounds of the limitations in the context of claim 1 is unclear giving the specified order of steps recited therein.
(c) Claims 21 and 23 are indefinite at the recitation of “DNMT1”, “DNMT5” and “TET” because abbreviations may have more than one meaning in the art. It is suggested inserting the full name of the abbreviation into the claim(s).
(d) Claim 31 is indefinite and lacks proper antecedent basis as the recitation of “administering or recommending administration of a chemotherapeutic agent to said subject based at least in part on said detecting same cells of said cancer” because no actual “detecting” step is recited in the claim 1 and no “cells of cancer” is recited or have been analyzed in the claim 1 from which the instant claim depends. Further it is unclear the metes and bounds of administering or recommending administration of a chemotherapeutic agent in the context of the claims.
(d) Claims 33 and 34 are indefinite at the recitation of “at least about” because a limiting definition has not been recited in the claims and thus the metes and bounds of the claimed limitations are unclear.
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
13. The claims 1-2, 5-9 and 16-39 are rejected under 35 USC 101 because the claimed invention is directed to judicial exceptions without significantly more. The claim(s) recite(s) both a law of nature and an abstract idea. The claims (s) do not include additional elements that are sufficient to amount to significantly more than the judicial exception. The rational for this finding is explained below:
M.P.E.P. § 2106. Regarding judicial exceptions, “[p]henomena of nature, though just discovered, mental processes, and abstract intellectual concepts are not patentable, as they are the basic tools of scientific and technological work.” Gottschalk v. Benson, 409 U.S. 63, 67 (1972); see also M.P.E.P. § 2106, part II.
The unpatentability of abstract ideas was recently confirmed by the U.S. Supreme Court. Alice Corp. Pty. Ltd. v. CLS Bank Int’l, 134 S. Ct. 2347, 2354 (2014); Bilski v. Kappos, 561 U.S. 593, 601 (2010).
The unpatentability of laws of nature was recently confirmed by the U.S. Supreme Court in Mayo Collaborative Servs. v. Prometheus Labs., Inc., 566 U.S. 66, 71 (2012). “[L]aws of nature, natural phenomena, and abstract ideas” are not patentable. Dia-mond v. Diehr, 450 U.S. 175, 185 (1981); see also Bilski v. Kappos, 561 U.S. at 601 (2010). The Supreme Court does acknowledge that it is possible “to transform an unpatentable law of nature,” but “one must do more than simply state the law of nature while adding the words ‘apply it.’” Mayo, 566 U.S. at 72 (quoting Gottschalk, 409 U.S. at 71–72). In Mayo, the Court found that “[i]f a law of nature is not patentable, the neither is a process reciting a law of nature, unless that process has additional features that provide practical assurance that the process is more than a drafting effort designed to monopolize the law of nature itself." 566 U.S. at 77. Additionally, “‘conventional or obvious’ ‘[pre]-solution activity’ is normally not sufficient to transform an unpatentable law of nature into a patent-eligible application of such a law.” Id. at 79 (quoting Flook, 437 U.S. at 590); see also Bilski, 561 U.S. at 593 (“[T]he prohibition against patenting abstract ideas ‘cannot be circumvented by’ . . . adding ‘insignificant post-solution activity’”) (quoting Diehr, 450 U.S. at 191–192).
The unpatentability of natural products was recently reconfirmed by the Supreme Court in Ass’n for Molecular Pathology v. Myriad Genetics, Inc., 133 S. Ct. 2107, 2116, (2013).
In Alice Corp., the Supreme Court reiterated the two-step test it devised in Mayo to determine patent eligibility for claims: “First, we determine whether the claims at issue are directed to one of those patent-ineligible concepts. If so, we then ask, ‘[w]hat else is there in the claims before us?’ To answer that question, we consider the elements of each claim both individually and ‘as an ordered combination’ to determine whether the additional elements ‘transform the nature of the claim’ into a patent-eligible application.” 134 S. Ct. at 2355 (citing and quoting 566 U.S. at 72-73, 76-78).
The following three inquiries are used to determine whether a claim is drawn to patent-eligible subject matter:
Step 1. Is the claim to a process, machine, manufacture, or composition of matter? Yes- the claims are clearly directed to a process.
The step 2 analysis is a two-part analysis, Step 2A and 2B.
Step 2A. Is the claim directed to a law of nature, a natural phenomenon, or an abstract idea (judicially recognized exceptions)?
Yes- The claims are directed to a natural phenomenon/law of nature and abstract ideas.
The natural phenomenon/law of nature is the correlation between the recited the identity of 5mC and 5hmc in cfDNA fragment sequences and location/abundance of 5mC and 5hmC in cfDNA fragment sequences and status of cancer. These correlations are naturally occurring or natural phenomenon and exist apart from any human action.
The claims further recite abstract ideas. The step of the obtaining sequencing reads and applying a trained classifier. These steps broadly encompass a general computer and/or mental activity of comparison of any number of sequences and trained classifier based on unrecited mathematical calculations, algorithms and software.
The Court has made clear if a claim is directed essentially to a method of calculating, using a mathematical formula, even if the solution is for a specific purpose, the claimed method is non-statutory. In other words, patenting abstract ideas cannot be circumvented by attempting to limit the use [the idea] to a particular technological environment. In the instant case, the programmed computer processor and product amounting therefrom are considered mere instructions to implement an abstract idea.
The claims also recite the step of recommending administration of a chemotherapeutic agent, a step of comparing differentially methylated regions in a genome and a step of generating a report indicating cancer status which are considered mental steps, such as talking to a person or reading information on a computer screen or paper.
Mental activities, data analysis, and mathematical analysis are all considered to be abstract ideas.
Step 2B. Does the claim recite additional elements that amount to significantly more than the judicial exception?
No-there are no elements or combination of elements in the claims that are sufficient to ensure that the claim as a whole amount to significantly more than the judicial exception:
In the instant case it is noted that all of the method steps recited are considered to be judicial exceptions themselves (see discussion above). Applicants cannot rely on a judicial exception to show that the claims as a whole amount to significantly more than a judicial exception.
The present claims do not require performing any steps that are not routine and conventional. For example, the claims do not require using novel reagents to identify 5mC or 5hmC. See Ariosa Diagnostics, Inc. v. Sequenom, Inc., F. Supp. 2d, 2013 WL 5863022, at *10 (N.D. Cal. Oct. 30, 2013) noting that "had the inventors of the [patent-in-suit] created an innovative method of performing DNA detection while searching for paternally inherited cffDNA, such as a new method of amplification or fractionation, those claims would be patentable.”
The art teaches methods of generating sequencing data, sequences from nucleic acid molecules, and using methylation differences are well known in the art. Mazloom et al. (US Patent Application 2018/0032671 Feb 1, 2018) teaches a method of capturing DNA molecules with biotinylated oligonucleotides and sequencing (para 124 and 136-139). Mazloom et al. teaches applying a trained classifier (para 194). Mazloom et al. teaches the biotinylated oligonucleotides can hybridize to methylated target genomic regions of size of 10Kb, 100 KB, 500KB, 1000KB, 5000 Kb or 10000kb in length wherein the fragments include sets of 100 sampled regions (para. 54 and 349).
Glezer et al (WO 2023/034814, March 2023, effective filing date September 2021) teaches method steps of sequencing one or more cytosine nucleobase, e.g., 5mC or 5hmC, and obtaining sequencing reads (see para. [0096], [0130]-[0160], 0257] and claims).
Gross et al (US 2020064727, March 3, 2022) and Kennedy et al (20190390253, December 19, 2019) teach method comprising applying trained classifier to classify the subject based on more characteristic of differentially methylated regions in cfDNA sequences and cancer type (See entire document of Gross et al. See Kennedy at paragraph [0024] – [0028]), [0036], [0098], [0179]), obtaining sequencing reads on cfDNA sample ([0144], [0162] – [0164]).
The various steps therein are not only naturally occurring in populations, but involve using routine statistical methods commonly applied in the scientific field.
In view of the foregoing, the claims 1-2, 5-9 and 16-39 are rejected under section 101 as being directed to non-statutory subject matter.
Claim interpretation
14. The claims are sufficiently broad and ambiguous as indicated above. Therefore, for the purpose of application of prior art, the claims are being given the broadest reasonable interpretation by the examiner.
Claim Rejections - 35 USC § 103
15. In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
16. The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
17. This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
18. Claim(s) 1-2, 5-9 and 16-26, 29-39 is/are rejected under 35 U.S.C. 103 as being unpatentable over view of Kennedy et al (US 20190390253, December 2019) in view of Gross et al (US 20220064737, effective filing date February 2020).
Regarding claim 1-2, 5-9, 16-25, 29-30, 33 and 34, Kennedy et al teach the following:
[0010] The disclosure further provides a method of analyzing a nucleic acid population comprising nucleic acids with different extents of modification. In some instances, the disclosure provides methods for screening for characteristics (e.g., 5′ methylcytosine) associated with a disease. The method comprises contacting the nucleic acid population with an agent (such as a methyl binding domain or protein) that preferentially binds to nucleic acids bearing the modification; separating a first pool of nucleic acids bound to the agent from a second pool of nucleic acids unbound to the agent, wherein the first pool of nucleic acids are overrepresented for the modification, and the nucleic acids in the second pool are underrepresented for the modification; linking the nucleic acids in the first pool and/or second pool to one or more nucleic acid tags that distinguish the nucleic acids in the first pool and the second pool to produce a population of tagged nucleic acids; amplifying the tagged nucleic acids, wherein the nucleic acids and the linked tags are amplified; assaying sequence data of the amplified nucleic acids and linked tags; decoding the tags to reveal whether the nucleic acids for which sequence data has been assayed were amplified from templates in the first or second pool.
[0011] In some embodiments the modification is binding of nucleic acids to a protein. In some embodiments, the protein is a histone or transcription factor. In some embodiments, the nucleic acid modification is a post-replication modification to a nucleotide. In some embodiments, the post-replication modification is 5-methylcytosine, and the extent of binding of the capture agent to nucleic acids increases with the extent of 5-methylcytosines in the nucleic acid. In some embodiments, the post-replication modification is 5-hydroxymethylcytosine, and the extent of binding of the agent to nucleic acid increases with the extent of 5-hydroxymethylcytosine in the nucleic acid. In some embodiments, the post-replication modification is 5-formylcytosine or 5-carboxylcytosine and the extent of binding of the agent increases with the extent of 5-formylcytosine or 5-carboxylcytosine in the nucleic acid.
[0026] In another aspect provided herein is a method, comprising: providing a population of nucleic acid molecules obtained from a bodily sample of a subject; fractionating the population of nucleic acid molecules based on one or more characteristics to generate a plurality of groups of nucleic acid molecules, differentially tagging nucleic acid molecules in the plurality of groups to distinguish the nucleic acid molecules in each of the plurality of groups from one another based on the one or more characteristics; sequencing the plurality of groups of nucleic acid molecules to generate sequence reads; containing sufficient data to generate relative information about nucleosome positioning, nucleosome modification, or binding DNA-protein interaction for each of the plurality of groups of nucleic acid molecules. In one embodiment the method further comprises analyzing the sequence reads to generate relative information about nucleosome positioning, nucleosome modification, or binding DNA-protein interaction for each of the plurality of groups of nucleic acid molecules. In another embodiment the method further comprises using a trained classifier to classify the subject based on the one or more characteristics. In another embodiment the one or more characteristics comprise a quantitative characteristic of the mapped reads. In an embodiment the method further comprises using a trained classifier to classify the subject based on the one or more characteristics. In another embodiment the trained classifier classifies the one or more characteristics as associated with a type of cancer in the subject. In another embodiment the one or more characteristics are indicative of gene expression or status of a disease. In another embodiment the nucleic acid molecules are cell-free DNA (“cfDNA”). In another embodiment the one or more characteristic is a cancer marker.
[0098] Cancer can be indicated by epigenetic variations, such as methylation. Examples of methylation changes in cancer include local gains of DNA methylation in the CpG islands at the transcription start site (TSS) of genes involved in normal growth control, DNA repair, cell cycle regulation, and/or cell differentiation. This hypermethylation can be associated with an aberrant loss of transcriptional capacity of involved genes and occurs at least as frequently as point mutations and deletions as a cause of altered gene expression. DNA methylation profiling can be used to detect regions with different extents of methylation (“differentially methylated regions” or “DMRs”) of the genome that are altered during development or that are perturbed by disease, for example, cancer or any cancer-associated disease. The genome of cancer cells harbor imbalance in the above DNA methylation patterns, and therefore in functional packaging of the DNA.
[0100] A characteristic of nucleic acid molecules may be a modification, which may include various chemical or protein modifications (i.e. epigenetic modifications). Non-limiting examples of chemical modification may include, but are not limited to, covalent DNA modifications, including DNA methylation. In some embodiments, DNA methylation comprises addition of a methyl group to a cytosine at a CpG site (a cytosine followed by a guanine in a nucleic acid sequence). In some embodiments, DNA methylation comprises addition of a methyl group to adenine, such as in N.sup.6-methyladenine. In some embodiments, DNA methylation is 5-methylation (modification of the 5th carbon of the 6-carbon ring of cytosine). In some embodiments, 5-methylation comprises addition of a methyl group to the 5C position of the cytosine to create 5-methylcytosine (m5c). In some embodiments, methylation comprises a derivative of m5c. Derivatives of m5c include, but are not limited to, 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC), and 5-caryboxylcytosine (5-caC). In some embodiments, DNA methylation is 3C methylation (modification of the 3rd carbon of the 6-carbon ring of cytosine). In some embodiments, 3C methylation comprises addition of a methyl group to the 3C position of the cytosine to generate 3-methylcytosine (3mC). DNA methylation is critical for normal development and abnormality in methylation may disrupt epigenetic regulation. The disruption, e.g., repression, in epigenetic regulation may cause diseases, such as cancer. Promoter methylation in DNA may be indicative of cancer.
[0101] Protein modifications include binding to components of chromatin, particularly histones including modified forms thereof, and binding to other proteins, such as proteins involved in replication or transcription. The disclosure provides methods of processing and analyzing nucleic acids with different extents of modification, such that the nature of their original modification is correlated with a nucleic acid tag and can be decoded by sequencing the tag when nucleic acids are analyzed. Genetic variation of sample nucleic acid modifications can then be associated with the extent of modification (epigenetic variation) of that nucleic acid in the original sample.
[0162] Bisulfite-based sequencing and variants thereof provides a means of determining the methylation pattern of a nucleic acid. In some embodiments, determining the methylation pattern comprises distinguishing 5-methylcytosine (5mC) from non-methylated cytosine. In some embodiments, determining methylation pattern comprises distinguishing N.sup.6-methyladenine from non-methylated adenine. In some embodiments, determining the methylation pattern comprises distinguishing 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) from non-methylated cytosine. Examples of bisulfite sequencing include, but are not limited to oxidative bisulfite sequencing (OX-BS-seq), Tet-assisted bisulfite sequencing (TAB-seq), and reduced bisulfite sequencing (redBS-seq).
[0163] Oxidative bisulfite sequencing (OX-BS-seq) is used to distinguish between 5mC and 5hmC, by first converting the 5hmC to 5fC, and then proceeding with bisulfite sequencing as previously described. Tet-assisted bisulfite sequencing (TAB-seq) can also be used to distinguish 5mc and 5hmC. In TAB-seq, 5hmC is protected by glucosylation. A Tet enzyme is then used to convert 5mC to 5caC before proceeding with bisulfite sequencing, as previously described. Reduced bisulfite sequencing is used to distinguish 5fC from modified cytosines.
[0164] Generally, in bisulfite sequencing, a nucleic acid sample is divided into two aliquots and one aliquot is treated with bisulfite. The bisulfite converts native cytosine and certain modified cytosine nucleotides (e.g. 5-formylcytosine or 5-carboxylcytosine) to uracil whereas other modified cytosines (e.g., 5-methylcytosine, 5-hydroxylmethylcystosine) are not converted. Comparison of nucleic acid sequences of molecules from the two aliquots indicates which cytosines were and were not converted to uracils. Consequently, cytosines which were and were not modified can be determined. The initial splitting of the sample into two aliquots is disadvantageous for samples containing only small amounts of nucleic acids, and/or composed of heterogeneous cell/tissue origins such as bodily fluids containing cell-free DNA.
[0183] Cell-free nucleic acids can be isolated from bodily fluids through a fractionation or partitioning step in which cell-free nucleic acids, as found in solution, are separated from intact cells and other non-soluble components of the bodily fluid.
With regards to the limitations directed to the hair-linked strands, Kennedy teaches the following: [0116] Double-stranded nucleic acids can be differentially labelled by ligation to at least partially double stranded adapters. Typically, the double-stranded nucleic acids are ligated to such adapters at both ends. Either or both of such adapters can include a nucleic acid tag. If two adapters each having a tag are linked to the respective ends of a nucleic acid, the tag combination can function as an identifier. Single-stranded DNA or RNA molecules do not ligate to a significant extent to double-stranded ends of adapters and so do not receive a nucleic acid tag. The double-stranded adapters can be fully double-stranded or partially double-stranded as is the case for Y-shaped adapters or hairpin adapters. Exemplary sequences for Y-shaped adapters are shown below.
[0125] FIG. 2 shows a further exemplary scheme starting with a sample including double-stranded DNA, single-stranded DNA and single-stranded RNA, with a simplified workflow, most notably obviating a 5′DNA phosphorylation step. The double-stranded DNA in the sample is first ligated to hairpin adapters including nucleic acid tags. The sample is then 5′DNA dephosphorylated and RNA is then converted to cDNA and also ligated to different tags. Single-stranded DNA is then processed similarly to as in FIG. 1. In some embodiments, hairpin adapters can be cleaved into two strands prior to library amplification.
[0129] In some embodiments, the first form of nucleic acids that is differentially tagged is double-stranded nucleic acids, and the tagging is performed by attaching hairpin adaptors to double-stranded nucleic acids.
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With regards to the addition of glucose, Kennedy et al teach in the example 14 pecific glucosylation of 5-hmC is accomplished by following the protocol of the highly active 5-hmC glucosyltransferase enzyme from Zymo Research (zymoresearch.com/epigenetics/dna-hydroxymethylation/5-hmc-glycosyltransferase). J-Binding Protein-1 (JBP-1) specifically binds to glucosylated DNA with high affinity, allowing 5-hmC levels to be determined by JBP-1-based enrichment. Additionally, glucosylation of 5-hmC alters the digestion of DNA by several restriction enzymes, and therefore digestion patterns of 5-hmC-glucosylated DNA can be used to assess DNA hydroxymethylation status.
With regards to the limitations concerning DNMT1, Kennedy et al teach in example 11 and Fig 19 methylation profiling of DNMT1 gene and its implication in cancer monitoring (para. [0076], [0345], [0346]).
While Kennedy et al teach multiple embodiments of the instant invention, including wherein the method disclosed therein encompass trained classifiers that classifies one or more characteristics o as associated with a type of cancer in a subject in cell free DNA (see citations above esp. [0026] – [0027]), Kennedy et al do not expressly teach deamination of the double-stranded hairpin cfDNA fragments,, wherein the methodology encompass AOIBEC enzyme or helicase enzyme or wherein the method encompassing administering a chemotherapeutic agent or the capability of the trained classifier.
Regarding claims 1-2, 26, 29-30, 31, 32, 36-39, Gross et al method of processing cell-free DNA (cfDNA) from a subject having or suspected of having a cancer, comprising:(a) obtaining cfDNA fragment sequences from the subject, (b) obtaining sequencing reads from said cfDNA fragment sequences that identify: (i) said cfDNA fragment sequences; (ii) 5-methylcytosine (5mC) in said cfDNA fragment sequences; and (iii) 5-hydroxymethylcytosine (5hmC) in said cfDNA fragment sequences; and (c) applying a trained classifier to said sequencing reads thereby determining a cancer status, wherein said trained classifier has been trained indicating cancer status (see para, [0023] – [0028], [0030] – [0031], [0091], [0118] –[0126], [0153] – [0165], [0186] (see also entire document, including Figures, examples and claims). Gross et al teach the following:
[0028] In some embodiments, the trained classifier determines a presence or absence of cancer and, if the classifier determines a presence of cancer, the classifier determines a cancer type. In some embodiments, the subject is determined to have a cancer and the specificity is at least 0.990. In some embodiments, the ratio of the likelihood of accurately determining a hematological disorder to the likelihood of inaccurately determining a solid tumor is at least 25:1 or at least 50:1. In some embodiments, the ratio of the likelihood of accurately determining a hematological disorder to the likelihood of inaccurately determining a hematological disorder is at least 8, at least 12:1, or at least 16:1. In some embodiments, the likelihood of accurately determining a cancer type is at least 80%, at least 85%, or at least 89%. In some embodiments, the cancer is a stage I cancer and the likelihood of accurately determining a cancer type is at least 65%, at least 70%, at least 75%, or at least 80%.
Gross et al teach sequence reads from at least 200 target genomic regions from at least 500, 1000 or more differentially methylated regions [[0008], [0010] Gross teach deaminating cfDNA fragments comprising contacting the fragments with APOBEC ([0106]. Gross et al teach further comprising administering a chemotherapeutic agent to the subject based on cancer cell detection ([0017], [0038] and [0039] and finally, wherein trained classifier determines the presence or absence of cancer or a cancer type by: (i) generating a set of features for the sample, wherein each feature in the set of features comprises a numerical value; (ii) inputting the set of features into the classifier, wherein the classifier comprises a multinomial classifier; (iii) based on the set of features, determining, at the classifier, a set of probability scores, wherein the set of probability scores comprises one probability score per cancer type class and per non-cancer type class; and (iv) thresholding the set of probability scores based on one or more values determined during training of the classifier to determine a final cancer classification of the sample. In some embodiments, the set of features comprises a set of binarized features. In some embodiments, the numerical value comprises a single binary value. In some embodiments, the multinomial classifier comprises a multinomial logistic regression ensemble trained to predict a source tissue for the cancer. In some embodiments, the method further comprises determining the final cancer classification based on a top-two probability score differential relative to a minimum value, wherein the minimum value corresponds to a predefined percentage of training cancer samples that had been assigned the correct cancer type as their highest score during training of the classifier. In some embodiments, (i) in accordance with a determination that the top-two probability score differential exceeds the minimum value, assign a cancer label corresponding to the highest probability score determined by the classifier as the final cancer classification; and (ii) in accordance with a determination that the top-two probability score differential does not exceed the minimum value, assigning an indeterminate cancer label as the final cancer classification [0028]. Gross teach that the method allows enrichment for DNA molecule or fragments identified as informative for detecting a disease wherein the method allows for lowering the detection needs and cost
Gross et al teach that an advantage of the clamed invention is that the method disclosed herein allows enriching for DNA molecule or fragments identified as informative for detecting a disease, lowering the detection needs and sequencing cost [0108].
It would have been obvious to one of ordinary skill in the art at the time of the effecting filing date of the claimed invention to have been motivated to have modified the method of Kennedy to that of Gross since they are of similar scope and utilize overlapping methodologies for detecting cancer. The ordinary artisan would have been motivated to do so for the benefit of improved means of diagnosing and/or monitoring cancer with increased efficiency and lower cost as suggested by the teachings of Kennedy and Gross.
Conclusion
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/CYNTHIA B WILDER/ Primary Examiner, Art Unit 1681
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