DETAILED ACTION
Applicant's Remarks, filed 01/28/2025, have been fully considered. The following rejections and/or objections are either reiterated or newly applied in view of instant application amendments. They constitute the complete set presently being applied to the instant application. Herein, "the previous Office action" refers to the 07/31/2024 Non-Final Rejection.
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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 .
Election/Restrictions
Applicant’s election with traverse of Species IA, IIB and IIIA in the reply filed 04/17/2024 is acknowledged.
Election was made with traverse in the Applicant's remarks, filed 04/17/2024.
The Applicant contends that Species Group I.B (claims 15- 16) be considered with Species I.A (claims 12-13). Examiner withdraws the Species I.B election requirement.
Claims 17, 19, 22, and 26-28 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected species, there being no allowable generic or linking claim.
Claim Status
Claims 2, 4-7, and 9-31 are currently pending.
Claims 17, 19, 22, and 26-28 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected species, there being no allowable generic or linking claim. Election was made with traverse in the Applicant's remarks, filed 04/17/2024.
Claims 3 and 8 are newly canceled
Claims 2, 4-7, 9-16, 18, 20-21, 23-25, and 29-31 are under examination herein.
Claims 2, 4-7, 9-16,18,20-21,23-25 and 29-31 are rejected
Priority
As previously discussed, domestic Priority data as claimed by applicant:
This application is a CON of 16/389,753, filed 04/19/2019-now US Patent No. 10706957,
which is a CON of 14/495,791, filed 09/24/2014 PAT 10392666,
which is a CON of PCT/AU2013/001088, filed 09/20/2013,
which claims benefit of 61/830,571, filed 06/03/2013,
and is a CIP of 13/842,209, filed 03/15/2013-now US Patent No. 9732390,
which claims benefit of 61/703,512 09/20/2012.
Claim 7 recites treating the cell-free DNA molecules with Tet-assisted bisulfite conversion or oxidative bisulfite sequencing. This limitation is not support by the ‘512, ‘209 or ‘571 applications. Claim 7 is not granted the claim to the benefit of priority to the ‘512, ‘209 and ‘571 applications.
Claims 10 recite analyzing the hypermethylation of CpG islands, are not supported by the disclosures of ‘512, ‘209 and ‘571 applications, and are not granted the claim to the benefit of priority to the ‘512, ‘209 and ‘571 applications.
Claims 20-21, 23-25 recite limitations for normalizing the methylation level based on the measured sizes of the cell-free DNA molecules, and are not granted the claim to the benefit of priority to the ‘512, ‘209 and ‘571 applications.
Withdrawn Rejections/Objections
Rejections and/or objections not reiterated from previous office actions are hereby withdrawn in view of the amendments and Applicant’s remarks, filed 01/28/2025. Upon further consideration, new rejections are recited below.
All rejections of claim 3 and 8 are withdrawn; their cancellation moots the rejections.
The drawing and specification objections for color drawings, is withdrawn in view of replacement drawings.
The claim objections regarding claim 31 are withdrawn, in view of claim amendments.
The rejection for double patenting is withdrawn in view of properly filed Terminal Disclaimer, as discussed below.
The rejection under 35 U.S.C. 112(b) claims 2, 4-7, 9-16, 18, 20-21, 23-25, and 29-31, as being indefinite is withdrawn.
The rejection under 35 U.S.C. § 102(a)(1) of claims recited in the previous Office Action, is hereby withdrawn, in further view of instant application amendments.
The rejection under 35 U.S.C. § 103, is hereby withdrawn, recited in the previous Office Action, is hereby withdrawn, in further view of instant application amendments.
. Upon further consideration, new rejections are recited below.
Terminal Disclaimer
The terminal disclaimer filed on 01/28/2025 disclaiming the terminal portion of any patent granted on this application which would extend beyond the expiration date of US Application # 17/135,676 and Patent Nos. 10706957, 10392666, and 9732390, has been reviewed and is accepted. The terminal disclaimer has been recorded.
Information Disclosure Statement
The Information Disclosure Statements filed on 08/02/2024, 11/27/2024, 02/20/2025, 07/15/2025, and 09/26/2025, are in compliance with the provisions of 37 CFR 1.97 and has been considered. Signed copies of the IDS are included with this Office Action.
Drawings
The Drawings submitted 01/28/2025 are accepted by the Examiner.
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.
Claims 2, 4-7, 9-16, 18, 20-21, 23-25, and 29-31 are rejected under 35 U.S.C. 101 because the claimed invention is directed to natural phenomenon and abstract ideas without significantly more.
The instant rejection reflects the framework as outlined in the MPEP at 2106.04:
Framework with which to Evaluate Subject Matter Eligibility:
(1) Are the claims directed to a process, machine, manufacture, or composition of matter;
(2A) Prong One: Do the claims recite a judicially recognized exception, i.e. a law of nature, a natural phenomenon, or an abstract idea;
Prong Two: If the claims recite a judicial exception under Prong One, then is the judicial exception integrated into a practical application (Prong Two); and
(2B) If the claims do not integrate the judicial exception, do the claims provide an inventive concept.
Framework Analysis as Pertains to the Instant Claims:
With respect to step (1): yes, the claims are directed to a method for cancer evaluation with methylation analysis of cell-free DNA, therefore the answer is "yes".
With respect to step (2A)(1), the claims recite abstract ideas. To determine if the claims recite any concepts that equate to an abstract idea, law of nature, or natural phenomenon, MPEP at 2106.03 teaches abstract ideas include mathematical concepts (mathematical formulas or equations, mathematical relationships, and mathematical calculations), certain methods of organizing human activity, and mental processes (including procedures for collecting, observing, evaluating, and organizing information (see MPEP 2106.04(a)(2)). In the instant application, the claims recite the following limitations that equate to an abstract idea with mental steps and mathematical concepts.
With respect to the instant claims, under the step (2A)(1) evaluation, the claims are found herein to recite natural phenomenon (correlating methylation levels to cancer) and abstract ideas that fall into the grouping of mental processes (in particular steps for analyzing epigenetic information) and mathematical concepts (in particular mathematical relationships between epigenetic data).
The claims directing to natural phenomenon are as follows:
Natural phenomenon:
Claim 12: first classification of a level of cancer based on the first methylation level.
Claim 13: identifying a type of cancer associated with the human organism by comparing the first methylation level…
Claim 14: determining the first classification of the level of cancer based on the comparison.
The claims directing to abstract ideas are as follows:
Mental processes:
Claim 1: wherein analyzing each of the plurality of cell-free DNA molecules includes: determining a location of the cell-free DNA molecule in a genome of the human organism by mapping, via sequence reads, the cell-free DNA molecule to a part of a human genome;
Claim 2: determining whether the cell-free DNA molecule is methylated at one or more CpG sites of a plurality of CpG sites organized into a plurality of CpG islands… determining a respective number of cell-free DNA molecules at each of the plurality of CpG islands that are methylated…
Claim 10: CpG islands are determined to be hypermethylated CpG islands compared to a reference group.
Claim 12: determining a first classification of a level of cancer based on the first methylation level
Claim 13: wherein the first classification indicates that cancer exists for the human organism, the method further comprising identifying a type of cancer associated with the human organism by comparing the first methylation level to a corresponding value determined from other human organisms, wherein at least two of the other human organisms are identified as having different types of cancer.
Claim 14: wherein determining the first classification of the level of cancer based on the first methylation level comprises: comparing the first methylation level to a cutoff value; and determining the first classification of the level of cancer based on the comparison.
Claim 15: wherein the cutoff value is a specified distance from a reference methylation level established from another biological sample obtained from a healthy organism.
Claim 16: wherein the specified distance is a specified number of standard deviations from the reference methylation level.
Claim 18: wherein comparing the first methylation level to the cutoff value includes: determining a difference between the first methylation level and a reference methylation level; and comparing the difference to a threshold corresponding to the cutoff value.
Claim 23: for each of the plurality of cell-free DNA molecules, determining the size of the cell-free DNA molecule by comparing the pair of sequences to a reference genome; and selecting the cell-free DNA molecules having the first size.
Mathematical concepts:
Claim 2: calculating a first methylation level using the respective numbers of cell-free DNA molecules that are methylated;
Claim 11: wherein the first methylation level is calculated for a methylation profile.
Claim 18: wherein comparing the first methylation level to the cutoff value includes: determining a difference between the first methylation level and a reference methylation level; and comparing the difference to a threshold corresponding to the cutoff value.
Claim 20: measuring a size of the cell-free DNA molecules at the plurality of CpG sites, thereby obtaining measured sizes; and before comparing the first methylation level to the cutoff value, normalizing the first methylation level using cell-free DNA molecules having a first size.
Claim 21: wherein the first size is a range of lengths.
Claim 24: obtaining a functional relationship between size and methylation levels; and using the functional relationship to normalize the first methylation level, wherein the functional relationship provides scaling values corresponding to respective sizes.
Claim 25: computing an average size corresponding to the cell-free DNA molecules used to calculate the first methylation level; and multiplying the first methylation level by a corresponding scaling value.
Hence, the claims explicitly recite elements that, individually and in combination, constitute natural phenomenon and abstract ideas.
With respect to step (2A), under the broadest reasonable interpretation (BRI), the instant claims a method for cancer evaluation with methylation analysis of cell-free DNA. Instant claims are therefore directed to the judicial exceptions of abstract groupings, both mathematical (calculating…comparing the difference…measuring a size…cutoff values…normalizing…functional relationship…scaling values…computing an average size…multiplying…) and mental processes (determining methylation status…compared to reference…determining the size…selecting…) which can be performed with the human mind with pen and paper.
Because the claims do recite judicial exceptions, direction under step (2A)(2) provides that the claims must be examined further to determine whether they integrate the abstract ideas into a practical application (MPEP 2106.04(d). A claim can be said to integrate a judicial exception into a practical application when it applies, relies on, or uses the judicial exception in a manner that imposes a meaningful limit on the judicial exception. This is performed by analyzing the additional elements of the claim to determine if the abstract idea is integrated into a practical application (MPEP 2106.04(d).I.; MPEP 2106.05(a-h)). If the claim contains no additional elements beyond the judicial exception, the claim is said to fail to integrate into a practical application (MPEP 2106.04(d).III).
With respect to the instant recitations, the claims recite the following additional elements considered for practical application:
Claim 2: performing methylation-aware sequencing,. sequencing of at least 60,000 cell-free DNA molecules.
Claim 4: performing methylation-aware massively parallel sequencing.
Claim 5: methylation- aware sequencing generates at least 10 million reads.
Claim 6: treating the cell-free DNA molecules with sodium bisulfite; and performing sequencing of the treated cell-free DNA molecules.…
Claim 7: Tet-assisted bisulfite conversion or oxidative bisulfite sequencing for a detection of 5-hydroxymethylcytosine.
Claim 9: CpG sites are on a plurality of chromosomes.
Claim 15: wherein the cutoff value is a specified distance from a reference methylation level established from another biological sample obtained from a healthy organism.
Claim 16: wherein the specified distance is a specified number of standard deviations from the reference
Claim 21: wherein the first size is a range of lengths.
Claim 23: performing paired-end massively parallel sequencing of the plurality of cell-free DNA molecules to obtain pairs of sequences for each of the cell-free DNA molecules;
Claim 29: CpG sites are from disjointed regions
Claim 30: wherein the biological sample is a plasma sample or a serum sample.
Claim 31: non-transitory computer readable medium … a computer system…
Said steps that are “in addition” to the recited judicial exception in the instant claims represent those of mere instructions or field of use limitations (determining by mapping via sequence reads…methylation-aware sequencing…[paired-end] massively parallel sequencing…reads…bisulfite conversion/sequencing…specified distance/number…disjointed regions) to implement in the recited judicial exception and do not impart meaning to said recited judicial exception, such that is applied in a practical manner. Further with respect to the additional elements in the instant claims, these steps direct to mere data gathering and handling (to obtain methylation profiles…sequence reads…) to carry out the abstract idea without imposing any meaningful limitation on the abstract idea. Thereby these steps are insignificant extra-solutions activity steps and are insufficient to integrate an abstract idea into a practical application. (MPEP 2106.05(g).
Further steps herein directed to additional non-abstract elements of computer components (claim 31: non-transitory computer readable medium … a computer system…) do not describe any specific computational steps by which the “computer parts” perform or carry out the abstract idea, nor do they provide any details of how specific structures of the computer, such as the computer-readable recording media, are used to implement these functions. The claims state nothing more than generic computer elements used as a tool to perform the functions that constitute the abstract idea. Hence, these are mere instructions to apply the abstract idea using a computer, and therefore the claim does not integrate that abstract idea into a practical application. The courts have weighed in and consistently maintained that when, for example, a memory, display, processor, machine, etc.… are recited so generically (FIG.3, XIV: [0508-0513]) that they represent no more than mere instructions to apply the judicial exception on a computer, and these limitations may be viewed as nothing more than generally linking the use of the judicial exception to the technological environment of a computer. (see MPEP 2106.05(f)). None of the recited dependent claims recite additional elements which would integrate a judicial exception into a practical application.
As such, the claims are lastly evaluated using the step (2B) analysis, wherein it is determined that because the claims recite natural phenomenon and abstract ideas, and do not integrate that abstract ideas into a practical application, the claims also lack a specific inventive concept. The judicial exception alone cannot provide the inventive concept or the practical application and that the identification of whether the additional elements amount to such an inventive concept requires considering the additional elements individually and in combination to determine if they provide significantly more than the judicial exception. (MPEP 2106.05.A i-vi).
With respect to the instant claims, the additional elements of data gathering, instructions, and field of use limitations described above do not rise to the level of significantly more than the judicial exception. As directed in the Berkheimer memorandum of 19 April 2018 and set forth in the MPEP, determinations of whether or not additional elements (or a combination of additional elements) may provide significantly more and/or an inventive concept rests in whether or not the additional elements (or combination of elements) represents well-understood, routine, conventional activity. Said assessment is made by a factual determination stemming from a conclusion that an element (or combination of elements) is widely prevalent or in common use in the relevant industry, which is determined by either a citation to an express statement in the specification or to a statement made by an applicant during prosecution that demonstrates a well-understood, routine or conventional nature of the additional element(s); a citation to one or more of the court decisions as discussed in MPEP 2106(d)(II) as noting the well-understood, routine, conventional nature of the additional element(s); a citation to a publication that demonstrates the well-understood, routine, conventional nature of the additional element(s); and/or a statement that the examiner is taking official notice with respect to the well-understood, routine, conventional nature of the additional element(s).
With respect to the instant recitations, the claims recite the following additional elements considered for inventive concepts:
Claim 2: performing methylation-aware sequencing,. sequencing of at least 60,000 cell-free DNA molecules.
Claim 4: performing methylation-aware massively parallel sequencing.
Claim 5: methylation- aware sequencing generates at least 10 million reads.
Claim 6: treating the cell-free DNA molecules with sodium bisulfite; and performing sequencing of the treated cell-free DNA molecules.…
Claim 7: Tet-assisted bisulfite conversion or oxidative bisulfite sequencing for a detection of 5-hydroxymethylcytosine.
Claim 9: CpG sites are on a plurality of chromosomes.
Claim 15: wherein the cutoff value is a specified distance from a reference methylation level established from another biological sample obtained from a healthy organism.
Claim 16: wherein the specified distance is a specified number of standard deviations from the reference
Claim 21: wherein the first size is a range of lengths.
Claim 23: performing paired-end massively parallel sequencing of the plurality of cell-free DNA molecules to obtain pairs of sequences for each of the cell-free DNA molecules;
Claim 29: CpG sites are from disjointed regions
Claim 30: wherein the biological sample is a plasma sample or a serum sample.
Claim 31: non-transitory computer readable medium … a computer system…
These additional elements do not contribute significantly more to well-known and conventional steps to obtain genetic data, performed with routine laboratory equipment, and analyzed by one with ordinary skill in the art as of the effective filing date. There is no active step of DNA sequencing or methylation detection with quantitative methylation [0084-0085] and bisulfite techniques [0096-0097], which is unconventional. The instant claims and references cited in the specification recite steps known in the art by molecular biologists, for sequencing genetic and epigenetic data with massively parallel and bisulfite sequencing were limitations equating to well-understood, routine and conventional activities in:
Laird [2010: Nature Reviews Genetics, 11: 191-203; PTO 892 cited] discloses a review of principles and challenges in genome-wide DNA methylation analysis including a review of methylation aware assay techniques that enable the counting of individual methylated DNA molecules, including sequencing based methods with sodium bisulfite treatment [Abstract; Table 1; pgs. 192-197].
Booth et al. [May 18, 2012: Quantitative Sequencing of 5-Methycytosine and 5-Hydroxymethylcytosine at Single-Base Resolution, Science, 336 (6083): 934-937; PTO 892 cited] discloses quantitative techniques with methylation-aware sequencing include Tet-assisted bisulfite conversion [Abstract].
Korshunova et al. 2008 discloses an analysis of the DNA-methylation profiles of loci of four aberrantly methylated genomic regions at high frequency in correlation with breast cancer (CGI) in over 700,000 patient derived DNA fragments from cancer-free and breast carcinomas sera obtained via a massively parallel bisulphite sequencing [PTO 892 cited; p20 Col 1].
Kim et al. [2011: Deep sequencing reveals distinct patterns of DNA methylation in prostate cancer. Genome research 21.7: 1028-1041] mapped global CGI methylation patterns of prostate cancer, using MethylPlex–next-generation sequencing (M-NGS) and bisulfite conversion, to identify ~68,000 methylated regions per sample, especially of promoter regions for 2481 cancer-specific differentially methylated regions (DMRs) [Abstract, FIG 1, p1031 Col 2].
Varley KE et al. [2010: Bisulfite Patch PCR enables multiplexed sequencing of promoter methylation across cancer samples. Genome research, 20(9), 1279-1287] demonstrates tumor-specific aberrant DNA methylation in colon and breast cancer with next generation sequencing/bisulfite techniques, obtaining almost 100K reads aligned to bisulfite treated reference sequences, analyzing 50+ differentially methylated loci(CGI) across a large number of samples [p1281 Col 1, FIG 2A].
Data (sequences/reads, methylation status) are merely manipulated data to be used in the judicial exception. The additional elements do not comprise an inventive concept when considered individually or as an ordered combination, as evidenced by the cited references teaching the combination of elements as well as the individual elements themselves, that transforms the claimed judicial exception into a patent-eligible application of the judicial exception.
With respect to the instant claims, the steps (analyzing for methylation status and comparing with reference genomes of healthy and cancer subjects) and additional elements (sequencing techniques, non-transitory computer-readable medium) involving mathematical relationships and automated mental steps do not comprise an inventive concept when considered individually or as an ordered combination that transforms the claimed judicial exception into a patent-eligible application of the judicial exception. Therefore, the claims do not amount to significantly more than the judicial exception itself (Step 2B: No). As such, claims 2, 4-7, 9-16, 18, 20-21, 23-25, and 29-31 are not patent eligible.
Response to 101 Remarks
The Applicant's Remarks [p.10], filed 01/28/2025, regarding rejection under 35 U.S.C. §101 have been considered in light of instant application amendments, but are not persuasive, as recited above and taught by multiple references. Applicant asserted the step of "determining whether the cell-free DNA molecule is methylated at one or more CpG sites of a plurality of CpG sites organized into a plurality of CpG islands… provide an improvement to a technical field …, by CpG island methylation measurement for cancer screening and including claim 8 limitations are not routinely performed.
As discussed above, methylation aware sequencing and bisulfite treatment of “cell associated with cancer” were conventional in the art and merely restrict the application to a field of use/ technological environment, but are not unusual steps or unconventional combination of data gathering. These limitations are extra-solution activity for necessary data gathering (sequences/reads, methylation status) to be used in the judicial exception, whether the data set includes 1 cell-free DNA molecule or 60,000 cell-free DNA molecules. Dependent claims 4-7 detail data gathering, and claims 9 and 29-30 the data types. The additional elements do not comprise an inventive concept when considered individually or as an ordered combination, as evidenced by the cited references teaching the combination of elements as well as the individual elements themselves, that transforms the claimed judicial exception into a patent-eligible application of the judicial exception.
Claim Interpretation
Claim 2 recites the term “potentially” in “the biological sample including DNA molecules originating from normal cells and potentially from cells associated with cancer.” For the examination purposes, the term potentially is interpreted as indicating a possible property of said sample, e.g. cancer, as in “cells potentially associated with cancer.”
Claim Rejections - 35 USC § 103
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. 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 § 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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. § 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or
nonobviousness.
Note: citations from the instant application are italicized in the following section.
Instant claims 2, 4-6, 9-12, 20-21, 23, and 29-31 are rejected under 35 U.S.C. 103 as being unpatentable over Korshunova et al. [2008: Massively parallel bisulphite pyrosequencing reveals the molecular complexity of breast cancer-associated cytosine-methylation patterns obtained from tissue and serum DNA, 18:19–29; PTO 892 document, herein Korshunova]; in view of Kim JH et al. [2011: Deep sequencing reveals distinct patterns of DNA methylation in prostate cancer. Genome research 21.7:1028-1041; PTO 892 cited] and Varley KE et al. [2010: Bisulfite Patch PCR enables multiplexed sequencing of promoter methylation across cancer samples. Genome research, 20(9), 1279-1287; PTO 892 cited]
Regarding instant claim 2, instant application recites:
A method of analyzing a biological sample of a human organism, the biological sample comprising cell-free DNA originating from normal cells and potentially from cells associated with cancer, the method comprising:
analyzing a plurality of cell-free DNA molecules from the biological sample, wherein analyzing each of the plurality of cell-free DNA molecules includes: determining a location of the cell-free DNA molecule in a genome of the human organism by mapping, via sequence reads, the cell-free DNA molecule to a part of a human genome; and
determining whether the cell-free DNA molecule is methylated at one or more CpG sites of a plurality of CpG sites organized into a plurality of CpG islands, each of the plurality of CpG islands including more than one CpG site, wherein determining whether the cell-free DNA molecule is methylated at the one or more CpG sites comprises performing methylation- aware sequencing, wherein the plurality of cell-free DNA molecules is at least 60,000;
determining a respective number of the at least 60,000 cell-free DNA molecules at each of the plurality of CpG islands that are methylated; and calculating a first methylation level using the respective numbers of the at least 60,000 cell-free DNA molecules that are methylated at the plurality of CpG islands.
The prior art to Korshunova discloses a method of:
comprehensive methylation pattern (methylation level/profiles) analysis for breast cancer from human subjects’ biological samples/mixtures with cell-free DNA and/or tissue [Abstract: “collection of 50 patients”]
with steps including mapping cell-free DNA molecule in a reference genome [p. 20, col.2, para. 3: “mapping the results to particular patient samples using MethylMapper procedure”]
FIG 1: Green boxes denote the position of Takai and Jones-predicted CpG Islands/CPGI in the fourth track. The settings used to map the CpGI were G+C 55, O/E 0.60, and a min length of 200 bp (size of range of lengths) [p.21 FIG.1 caption]
providing DNA-methylation landscape for over 700,000 patient derived DNA fragments (wherein the plurality of cell-free DNA molecules is at least 60,000) [Abstract]. from sera (cell-free DNA) and cancer-free breast tissue (normal cells), infiltrating ductal breast carcinomas (levels of cancer) using a massively parallel bisulphite sequencing strategy. The targeted loci in the investigation were four genomic regions identified as aberrantly methylated at high frequency in correlation with breast cancer and provides information regarding the epigenetic pattern normally observed (threshold, reference methylation) in DNA circulating within the bloodstream [p20, Col 1, para last] (determining whether the cell-free DNA molecule is methylated at one or more CpG sites of a plurality of CpG sites organized into a plurality of CpG islands, each of the plurality of CpG islands including more than one CpG site, wherein determining whether the cell-free DNA molecule is methylated at the one or more CpG sites comprises performing methylation- aware sequencing, wherein the plurality of cell-free DNA molecules is at least 60,000;) and the cytosine-methylation topography (methylation profile at CPG sites in CPG islands) within the samples was analyzed for methylation pattern of each molecule and methylation density/percent of methylated residues from the total number of residues sequenced per molecule. The average methylation density of a region is the mean methylation occupancy per CG across a region/amplicon (methylation level). Within a region (CPG island/sites), the average methylation occupancy was calculated for each CG (CPG site) as a percent of the methylated C’s from the total number of molecules sequenced at that position [p23, Col 2, para 1] (calculating a first methylation level of a first chromosomal region based on the respective numbers of the at least 60,000 cell-free DNA molecules methylated at sites within the first chromosomal region).
However, Korshunova does not explicitly “determining a respective number of the at least 60,000 cell-free DNA molecules at each of the plurality of CpG islands that are methylated" where "each of the plurality of CpG islands including more than one CpG site.". Further, Korshunova does not specifically disclose "determining a location of the cell-free DNA molecule in a genome of the human organism by mapping, via sequence reads, the cell-free DNA molecule to a part of a human genome."
The prior art to Kim et al. mapped global CGI methylation patterns of prostate cancer, using MethylPlex–next-generation sequencing (M-NGS) and bisulfite conversion, to identify ~68,000 methylated regions per sample, especially of promoter regions for 2481 cancer-specific differentially methylated regions (DMRs) [Abstract, FIG 1, p1031 Col 2].
The prior art to Varley KE et al demonstrates tumor-specific aberrant DNA methylation in colon and breast cancer with next generation sequencing/bisulfite techniques, obtaining nearly 100,000 reads aligned to bisulfite treated reference sequences, analyzing 50+ differentially methylated loci(CGI) across a large number of samples [p1281 Col 1, FIG 2A].
Regarding instant claim 4, instant application recites:
claim 4: …methylation-aware massively parallel sequencing..
The prior art to Korshunova teaches using massively parallel bisulphite pyrosequencing for comprehensive methylation pattern analysis from breast cancer [Abstract], includes: treating the cell-free DNA molecules with sodium bisulfite; and performing sequencing of the treated cell-free DNA molecules [p27, Col 2: Preparation of samples for massively parallel bisulphite pyrosequencing].
Regarding instant claim 5, instant application recites:
wherein performing methylation- aware sequencing generates at least 10 million reads.
The prior art to Korshunova teaches the four loci studied for methylation analysis included more than 700,000 DNA fragments derived from more than 50 individuals [Abstract]. For inclusion in this study, the number of reads per sample per amplicon was set to be greater than 100 (Supplemental Table S2), making all occupancy and abundance calculations theoretically precise at or below the 1% level. Therefore, 700,000 cfDNA fragments when read per sample greater than 100, would result in at least 10 million reads (see Methods section) [p.22 Col 1 para 2].
Regarding instant claim 6, instant application recites:
treating the cell-free DNA molecules with sodium bisulfite; and performing sequencing of the treated cell-free DNA molecules.
The prior art to Korshunova teaches using massively parallel bisulphite pyrosequencing for comprehensive methylation pattern analysis from breast cancer [Abstract], includes: treating the cell-free DNA molecules with sodium bisulfite; and performing sequencing of the treated cell-free DNA molecules [p27, Col 2: Preparation of samples for massively parallel bisulphite pyrosequencing].
Regarding instant claim 9, instant application recites:
wherein the plurality of CpG sites are on a plurality of chromosomes.
The prior art to Korshunova discloses identifying a plurality of chromosomes/ chromosomal regions of a genome of the subject and CPG sites within chromosomal regions [Figure 1, A–D; p. 20, col. 1, last para., the targeted loci in the investigation were four genomic regions that we have recently identified as methylated at high frequency in correlation with breast cancer; “each chromosome is indicated in the top, left of each panel” (p.21 FIG.1 caption)]; calculating a region methylation level for each chromosomal region (p. 23, col. 2, para. 1, calculation of methylation density). Further, Korshunova discloses determining the location of the cell-free DNA molecule in the reference genome involves determining whether the location maps within chromosomal regions (figure 1, Green boxes denote the position of Takai and Jones-predicted CpG Islands in the fourth track. The settings used to map the CpGI were G+C 55, O/E 0.60, and a min length of 200 bp. Within the fifth track, aqua boxes denote the position of the sequencing amplicons used in the 454 experiments. The last track (yellow boxes) indicates the position of the qPCR assay used to establish the methylation status of the biomarkers).
Regarding instant claim 10, instant application recites:
wherein the plurality of CpG islands are determined to be hypermethylated CpG islands compared to a reference group.
The prior art to Korshunova teaches the targeted loci were four genomic regions identified as aberrantly methylated at high frequency (hypermethylated) in correlation with breast cancer and provides information regarding the epigenetic pattern normally observed (threshold, compared to a reference group) in DNA circulating within the bloodstream [p20, Col 1, para last].
Regarding instant claim 11, instant application recites:
wherein the first methylation level is calculated for a methylation profile.
The prior art to Korshunova teaches the cytosine-methylation topography (methylation profile) within the samples was analyzed for methylation pattern of each molecule and methylation density/percent of methylated residues from the total number of residues sequenced per molecule. The average methylation density of a region is the mean methylation occupancy per CG across a region/amplicon (methylation level). Within a region (CPG island/sites), the average methylation occupancy was calculated for each CG (CPG site) as a percent of the methylated C’s from the total number of molecules sequenced at that position [p23, Col 2, para 1].
Regarding instant claim 12, instant application recites:
determining a first classification of a level of cancer based on the first methylation level (instant claim 12)
The prior art to Korshunova teaches DNA-methylation analysis for over 700,000 patient derived DNA fragments from sera (cell-free DNA) and cancer-free breast tissue (normal cells), infiltrating ductal breast carcinomas (level of cancer). The targeted loci in the investigation were four genomic regions identified as aberrantly methylated (methylation level) at high frequency in correlation with breast cancer and provides information regarding the epigenetic pattern normally observed (threshold, reference methylation) in DNA circulating within the bloodstream [p20, Col 1, para last]. Additionally, Korshunova determines the methylation landscape of DNA derived from diseased and non-diseased (normal cells) breast tissue, breast tumors (stage II infiltrating ductal carcinoma) and 11 histologically normal breast tissues and serum samples (cell-free DNA) from 21 breast cancer patients (stages I–IIIB) and 21 cancer-free individuals [Table 1, p22 Col 1 para 3]).
Regarding instant claims 20-21, instant application recites:
measuring a size of the cell-free DNA molecules at the plurality of CpG sites, thereby obtaining measured sizes; and before comparing the first methylation level to the cutoff value, normalizing the first methylation level using cell-free DNA molecules having a first size (instant claim 20).
wherein the first size is a range of lengths (instant claim 21).
The prior art to Korshunova teaches subdividing methylome characterized genomic loci into two to four regions of 100–200 bp (measuring…having a first size) which is consistent with highly fragmented cfDNA size found in sera (p.21 FIG 1 caption: “the settings used to map the CpGI…a min length of 200 bp”; Supplemental Table S1) and provides a reference control (normalizing) when sequencing breast tissue-derived molecular libraries [p.20 Col 2 para 2].
Regarding instant claim 23, instant application recites:
performing paired-end massively parallel sequencing of the plurality of cell-free DNA molecules to obtain pairs of sequences for each of the cell-free DNA molecules;
for each of the plurality of cell-free DNA molecules, determining the size of the cell-free DNA molecule by comparing the pair of sequences to a reference genome; and
selecting the cell-free DNA molecules having the first size..
The prior art to Korshunova teaches:
subdividing methylome characterized genomic loci into two to four regions of 100–200 bp (measuring…having a first size) which is consistent with highly fragmented cfDNA size found in sera (p.21 FIG 1 caption: “the settings used to map the CpGI…a min length of 200 bp”; Supplemental Table S1) and provides a reference control (normalizing) when sequencing breast tissue-derived molecular libraries [p.20 Col 2 para 2].
The locus-specific tag-bearing PCR products were amplified from bisulphate-modified DNA, purified, quantified individually, mixed in equimolar amounts, and sequenced using the 454 pyrosequencing platform. [p.20 Col 2 para 4].
Regarding instant claim 29, instant application recites:
wherein the plurality of CpG sites are from disjointed regions separated from each other.
The prior art to Korshunova discloses identifying a plurality of chromosomes/chromosomal regions (disjointed regions) of a genome of the subject and CPG sites within chromosomal regions [Figure 1, A–D; p. 20, col. 1, last para., the targeted loci in the investigation were four genomic regions that we have recently identified as methylated (CpG sites) at high frequency in correlation with breast cancer; “each chromosome is indicated in the top, left of each panel“ [p.21 FIG.1 caption].
Regarding instant claim 30, instant application recites:
wherein the biological sample is a plasma sample or a serum sample.
The prior art to Korshunova teaches providing DNA-methylation landscape for over 700,000 patient derived DNA fragments (cell-free DNA) from serum samples [p20, Col 1, para last].
Regarding instant claim 31, instant application recites:
A non-transitory computer readable medium comprising a plurality of instructions capable of execution by a computer system, that when so executed control the computer system to perform the method of claim 2.
The prior art to Korshunova discloses carrying out Statistical analyses using SAS (SAS Institute) and R (R Foundation for Statistical Computing) software. It is inherent that these software products are stored on a non-transitory computer readable and control a computer system when executed by the computer.
Therefore, it would have been obvious to someone of ordinary skill in the art, before the effective filing date of the claimed invention, to have modified Korshunova massively parallel bisulfite sequencing in DNA methylation profiling to incorporate Kim’s expansive analysis of ~68,000 methylated regions across multiple DMR/CGIs [Kim at Abstract, FIG 1, p1031 Col 2] and Varley’s use of 100,000 reads aligned to bisulfite treated reference sequences, analyzing 50+ differentially methylated loci(CGI) [Varley at p1281 Col 1, FIG 2A]. Combining these prior art elements would have been obvious in order to achieve a more comprehensive human cancer methylome map, as Kim teaches global CGI methylation is significantly increased at promoters sites between benign and cancerous prostate tissues [Kim at Abstract]. One of ordinary skill in the art would predict that analyzing a larger number of DNA fragments with a human genome reference library to characterize a plurality of CGI sites could be readily added to Korshunova’s breast cancer methylome profiling with reasonable expectation of success, as this permits identification of multiple gene promoters that exhibit tumor-specific DNA methylation defects that occur frequently in different cancer types, not only breast cancer but also colon cancer [Varley at Abstract] and prostate cancer [Kim at Abstract]. The invention is therefore prima facie obvious.
B. Claim 7 is rejected under 35 U.S.C. 103 as being unpatentable over Korshunova in view of Kim and Varley, as applied to claims 2 and 6 above, and in view of Yu et al. (Yu et al. Nature Protocols 2012, Vol 7, No 12, p. 2159-2170; PTO 892 document, herein Yu).
Regarding instant claim 7, instant application recites:
with the sodium bisulfite is part of Tet-assisted bisulfite conversion or oxidative bisulfite sequencing for a detection of 5-hydroxymethylcytosine.
Korshunova discloses the limitations as pertain to claims 2 and 6, as recited above.
The prior art to Korshunova further discloses using massively parallel bisulphite pyrosequencing for comprehensive methylation pattern analysis from breast cancer [Abstract], includes: treating the cell-free DNA molecules with sodium bisulfite; and performing sequencing of the treated cell-free DNA molecules [p27, Col 2: Preparation of samples for massively parallel bisulphite pyrosequencing].
However, Korshunova is silent to sodium bisulfite as part of Tet-assisted bisulfite conversion or oxidative bisulfite sequencing for detection of 5-hydroxymethylcytosine in claim 7. The prior art to Yu discloses a modified bisulfite sequencing method, called Tet-assisted bisulfite sequencing (abstract; pg. 2159, col. 1, para. 1 to col. 2, para. 3).
Therefore, it would have been obvious to someone of ordinary skill in the art, before the effective filing date of the claimed invention, to have modified Korshunova in view of Kim and Varley s traditional bisulfite sequencing to incorporate Yu’s Tet-assisted bisulfite sequencing method for differentiation between 5-methylcytosine and 5-hydroxymethylcytosine [p 2159 Col 1 para 3 to Col 2 para 1].. Combining these prior art elements would have been obvious because Tet-assisted bisulfite methods improve methylome analysis sensitivity/specificity and defines other biological functions through additional methylation forms [instant specification [0096]). One of ordinary skill in the art would predict that the Tet-assisted bisulfite sequencing method taught by Yu could be readily added to Korshunova with reasonable expectation of success, since Yu teaches this method is compatible with sequencing techniques [p 2159 Col 2 para 4]. The invention is therefore prima facie obvious.
C. Claims 13 and 24-25 are rejected under 35 U.S.C. 103 as being unpatentable over Korshunova in view of Kim and Varley, as applied to claims 2, 12, 14, and 20 above, in view of Christensen et al. (US 2011/0028333 A1; PTO 892 cited, herein Christensen).
Regarding instant claim 13, instant application recites:
identifying a type of cancer associated with the human organism by comparing the first methylation level to a corresponding value determined from other human organisms, wherein at least two of the other human organisms are identified as having different types of cancer.
Korshunova disclose the limitations as pertain to claims 2 and 12 as recited above. The prior art to Korshunova further teaches:
DNA-methylation analysis for over 700,000 patient derived DNA fragments from sera (cell-free DNA) and cancer-free breast tissue (normal cells), infiltrating ductal breast carcinomas (level of cancer) The targeted loci in the investigation were four genomic regions identified as aberrantly methylated at high frequency in correlation with breast cancer and provides information regarding the epigenetic pattern normally observed (threshold, reference methylation) in DNA circulating within the bloodstream [p20, Col 1, para last]. Additionally, Korshunova determines the methylation landscape of DNA derived from diseased and non-diseased (normal cells) breast tissue, breast tumors (stage II infiltrating ductal carcinoma) and 11 histologically normal breast tissues and serum samples (cell-free DNA) from 21 breast cancer patients (stages I–IIIB) and 21 cancer-free individuals [Table 1, p22 Col 1 para 3]).
However, Korshunova does not teach classification of different cancers.
The prior art to Christensen teaches a method for cancer differentiation from cfDNA based on the methylation levels (Christensen at [0011-0030; 0051-0080]). Christensen further teach that this method includes mixture modeling to either Tumor or Normal control (reference) methylation data and calculation of methylation levels for each CpG locus with BeadStudio Methylation software (Christensen at [0056; 0064]) to establish cancer comparison and classification metrics against reference groups (Christensen at [0011-0024; 0064-0067]) for lung adenocarcinoma and pleural mesothelioma (different types of cancer) (Christensen at [0011-0024; 0068-0089]). Christensen further discloses that the methylation level is calculated by dividing the number of methylated regions by the total number of regions (Christensen at [0064]).
Regarding instant claims 24-25, instant application recites:
wherein normalizing the first methylation level using the cell-free DNA molecules having the first size includes: obtaining a functional relationship between size and methylation levels; and using the functional relationship to normalize the first methylation level, wherein the functional relationship provides scaling values corresponding to respective sizes (instant claim 24).
computing an average size corresponding to the cell-free DNA molecules used to calculate the first methylation level; and multiplying the first methylation level by a corresponding scaling value (instant claim 25).
The limitations of claims 2, 12, 14, and 20 have been taught by above rejection.
The prior art to Korshunova teaches:
100–200 bp (average size) short read lengths are ideally suited for the study of serum DNA as it is highly fragmented in sera [Korshunova at p.20 Col 2 para 2].
subdividing methylome characterized genomic loci into two to four regions of 100–200 bp (measuring…having a first size) which is consistent with highly fragmented cfDNA size found in sera (p.21 FIG 1 caption: “the settings used to map the CpGI…a min length of 200 bp”; Supplemental Table S1) and provides a reference control (normalizing) when sequencing breast tissue-derived molecular libraries [Korshunova at p.20 Col 2 para 2].
However, Korshunova does not teach functional relationships and scaling values.
The prior art to Christensen teaches:
Scaling parameters (scaling values) to test CpG loci associations between sample type (cfDNA vs tissue or sample DNA fragment sizes), or covariates such as age or gender and methylation at individual CpG loci were tested with a generalized linear model (GLM). The beta-distribution of average beta values was accounted for with a quasi-binomial logit link (functional relationships) with an estimated scale parameter (scaling values) constraining the mean between 0 and 1, in a manner similar to that described by Hsuing et al. CpG loci where an a priori hypothesis existed were tested independently. In contrast, array-wide scanning for CpG loci associations with sample type or covariate used false discovery rate correction and Q-values computed by the q-value package in R (Christensen at [0065]).
Therefore, it would have been obvious to someone of ordinary skill in the art, before the effective filing date of the claimed invention, to have modified Korshunova in view of Kim and Varley’s size-based cfDNA methylation analysis of cancerous versus healthy samples, to incorporate Christensen’s methylome application to multiple different cancers and sample scaling variables. Combining these prior art elements would have been obvious because methylome analysis can further noninvasively differentiate cancer types/conditions when more methylation profiles are characterized with associations from uniquely sized cfDNA fragments [instant specification [0096]). One of ordinary skill in the art would predict a reasonable expectation of success, as both Korshunova and Christensen teach cancer-related methylome testing in cfDNA. The invention is therefore prima facie obvious.
D. Claims 14-16 and 18 are rejected under 35 U.S.C. 103 as being unpatentable over Korshunova in view of Kim and Varley, as applied to claim 2 and 12 above, in view of Fackler et al. (2004) Quantitative Multiplex Methylation-Specific PCR Assay for the Detection of Promoter Hypermethylation in Multiple Genes in Breast Cancer. Cancer Research 64: 4442–4452; PTO 892 cited, herein Fackler).
Regarding instant claims 14-16 and 18, instant application recites:
wherein determining the first classification of the level of cancer based on the first methylation level comprises: comparing the first methylation level to a cutoff value; and determining the first classification of the level of cancer based on the comparison (instant claim 14).
wherein the cutoff value is a specified distance from a reference methylation level established from another biological sample obtained from a healthy organism (instant claim 15).
wherein the specified distance is a specified number of standard deviations from the reference methylation level (instant claim 16).
wherein comparing the first methylation level to the cutoff value includes: determining a difference between the first methylation level and a reference methylation level; and comparing the difference to a threshold corresponding to the cutoff value (instant claim 18).
Korshunova disclose the limitations as pertain to claims 2 and 12, as recited above. However, Korshunova does not teach cutoff methylation values, and additionally, Korshunova brings into question the general utility of cutoff/threshold values for methylation profiles [p26 Col 2 para 1: “many serum methylation studies and related techniques typically suffer from specificity issues/false positives when using predetermined thresholds (threshold…cutoff values), because the complexity of the methylation patterns observed suggests that diagnostic cutoff values will likely vary substantially between patient cohorts, making consistent application of the same (or a standardized) cutoff clinically less feasible in serum testing.”]
The prior art to Fackler teaches a methylation level cutoff for each gene at the 10th percentile of the population (threshold, as defined by instant specification [0297, 0317]), such that 90% of reference/normal breast tissue would be at or below cutoffs (“cutoffs of 2% M for RASSF1A and HIN1, 0.5% M for TWIST, and 0.2% M for Cyclin D2” in normal tissues) and values above cutoffs were “positive” for hypermethylation [p 4447 Col 2 para 1-2]. Reference cutoff values for cumulative methylation profiles determined from healthy subjects compared with those of cancer patients [Tables 5-6; p4448, Col 1-2] and comparing CpG island hypermethylation of chromosomal promoter regions/loci in breast samples for a statistically significant difference in Mann-Whitney testing [FIG 7, p4447 Col 2 para 1-2] (specified distance/number of standard deviations from the reference/a normal sample, as defined in instant specification [0212]).
Therefore, it would have been obvious to someone of ordinary skill in the art, before the effective filing date of the claimed invention, to have modified Korshunova in view of Kim and Varley’ s cfDNA methylation analysis of cancerous conditions to incorporate Fackler’s methylation cutoffs. Combining these prior art elements would have been obvious because cfDNA detection of low vs. high methylation status based on methylation level cutoffs can be associated with healthy vs. cancerous classification noninvasively [Korshunova at p22 Col 1 para 3]). One of ordinary skill in the art would predict a reasonable expectation of success, as both Korshunova and Christensen teach cancer-related methylome testing in cfDNA. The invention is therefore prima facie obvious.
Response to 102/103 Remarks
The Applicant's Remarks [p.12-13], filed 01/28/2025, regarding prior art rejections have been considered in view of instant application amendments, but are not persuasive regarding limitations “determining a respective number of the at least 60,000 cell-free DNA molecules at each of the plurality of CpG islands that are methylated" where "each of the plurality of CpG islands including more than one CpG site…determining a location of the cell-free DNA molecule in a genome of the human organism by mapping, via sequence reads, the cell-free DNA molecule to a part of a human genome” as disclosed by Korshunova in view of Kim and Varley, as recited above.
Conclusion
No claims are allowed.
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/VR/
Examiner
Art Unit 1685
/MARY K ZEMAN/Primary Examiner, Art Unit 1686