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 .
Status of Claims
This case has been transferred to a new examiner for continued examination. Any further communications regarding this case may be directed to the contact information included in the conclusion of this office action.
Status of Claims
Applicant’s amendment filed 09/22/2025 is acknowledged. Claims 25, 27-40, 42-63, and 66-138 have been cancelled. Claims 1-24, 26, 41, and 64-65 are pending in the instant application and the subject of this non-final office action.
Election/Restrictions
Applicant’s election without traverse of Group I, claims 1-24, 26, 41, and 64-65 in the reply filed on 09/22/2025 is acknowledged.
Drawings
The drawings are objected to as failing to comply with 37 CFR 1.84(p)(5) because they include the following reference character(s) not mentioned in the description:
5502, 5504, and 5506 are missing. Paragraph [0289] recites 4802, 4804, and 4806 instead.
Corrected drawing sheets in compliance with 37 CFR 1.121(d), or amendment to the specification to add the reference character(s) in the description in compliance with 37 CFR 1.121(b) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
Specification
The use of the terms including “SMRT” and “SMRTbell”, each of which is a trade name or a mark used in commerce, has been noted in this application. Such terms should be accompanied by the generic terminology; furthermore, such terms should be capitalized wherever they appear or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the terms.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
Claim Objections
Claim 23 is objected to because of the following informalities:
Claim 23: The claim recites “a plurality…include the first…pattern”; it should be “includes”.
Appropriate correction is required.
Claim Interpretation
In evaluating the patentability of the claims presented in this application, claim terms have been given their broadest reasonable interpretation (BRI) consistent with the specification, as understood by one of ordinary skill in the art, as outlined in MPEP 2111.
Regarding claim 1, the claims recites “methylation-aware sequencing of a plurality of cell-free DNA molecules…wherein the plurality of cell-free DNA molecules have a first size range”.
Regarding the upper bound of the first size range, see the 112(b) rejection below.
Regarding the “methylation-aware sequencing”, this term is defined in para [0113] of the instant specification as “any sequencing method that allows one to ascertain the methylation status of a DNA molecule during a sequencing process, including, but not limited to bisulfite sequencing, or sequencing preceded by methylation-sensitive restriction enzyme digestion…or single molecule sequencing that allows elucidation of the methylation status
(e.g., without bisulfite sequencing).” Given that the exemplary sequencing methods allow for processing steps as part of the “sequencing”, the term has been interpreted broadly to encompass other such processing of nucleic acid molecules known in the art as part of a sequencing preparation.
Accordingly, the size range of cfDNA sequenced in the claim has been interpreted broadly to encompass at least the inherent lengths of cfDNA in a sample and fragmented lengths.
Regarding claim 17, the claim recites “each plurality of sites [of the first methylation pattern of a reference tissue] identifies a methylation index at the site”. The instant specification fails to recite a limiting definition for “methylation index” but offers that it “can refer to the proportion of DNA fragments showing methylation at the site” or that it “can be transformed into a binary value” (para [0106]). Therefore, the term was interpreted broadly.
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 1-24, 26, 41, and 64-65 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.
In analyzing the claims for compliance with the written description requirement of 35 U.S.C. 112, first paragraph, the written description guidelines note that with regard to genus/species situations, a “Satisfactory disclosure of a “representative number'' depends on whether one of skill in the art 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.”
Regarding claims 1-9, 12-24, 41, and 64-65, the claims are directed to a method of analyzing a biological sample comprising, for each of a sequence read obtained from methylation-aware sequencing of a plurality of cfDNA molecules from the biological sample, comparing a methylation pattern of methylation status at a set of sites on the sequencing read to a first reference methylation pattern, wherein the reference methylation pattern corresponds to a first tissue type and based on the comparison, determining a tissue classification of the sequence read, and further determining a classification of a disease of the biological sample based on the tissue classification of the sequence reads.
The claim have great breadth reading at least any biological sample type for any disease (aside from claims 10-11 and 26, which specify cancer).
Contrary to this, the disclosure recites determining the tissue of origin for plasma DNA for disease classification of HCC and CRC (e.g., para [0248-260], [0279], and Fig. 54). Thus, the disclosure teaches the disease cancer and the sample plasma.
While other biological samples are contemplated in para [0092] and diseases are contemplated in para [0116], a representative number, neither individually nor in combination, are reduced to practice or described in sufficient detail to show sufficient possession (e.g., necessary alterations of the method for diseases other than cancer, which has an inherent immune response that results in apoptosis and release of cfDNAs into the bloodstream).
Arockiaraj (Arockiaraj AI, et al. Methylation Data Processing Protocol and Comparison of Blood and Cerebral Spinal Fluid Following Aneurysmal Subarachnoid Hemorrhage. Front Genet. 2020 Jun 26;11:671) teaches, for example, that blood and CSF are not appropriate surrogates when studying Aneurysmal Subarachnoid Hemorrhage with methylation, as the sample types have different levels of within-CpG correlation (Abstract).
For this reason, there would be expected to be a high degree of variability in the genus claimed of any sample type for any disease and/or cancer of the types claimed, such that the single species demonstrated would not be sufficient for the skilled artisan to conclude that the applicant had possession of the enter genus of all biological samples combined with all possible diseases/each cancer type at the time of filing.
Thus, the claims fail to meet the written description requirements of 112(a).
Claim Rejections - 35 USC § 112(b)
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.
Claim 1-24, 26, 41, and 64-65 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.
Regarding claim 1, the claim recites “sequencing reads obtained from a…sequencing of a plurality of…DNA molecules…wherein the plurality of…DNA molecules have a first size range, and wherein an upper bound of the first size range is greater than 600 bases”.
Consistent with the art-accepted meaning of “upper bound” of a maximum size, the instant specification defines the upper bound as the “maximum size of the cell-free DNA molecule to be considered as a long DNA molecule” (para [0169]).
The claim is indefinite because the “maximum size” is indefinite towards higher numbers, i.e., the maximum size is set at infinity or the practical equivalent thereof for cfDNA; thus, no meaningful bounds have been provided for the first size range. Therefore, the metes and bounds cannot be established by one of ordinary skill in the art.
For the purposes of applying art, any range of cfDNA molecules is held to anticipate the limitation. See MPEP 2131.03.
Claims 2-24, 26, 41, and 64-65 are indefinite for depending from claim 1 or a dependent claim thereof and not rectifying the deficiency.
Regarding claim 17, the claim recites “each of the plurality of sites identifies a methylation index at the site”. There is insufficient antecedent basis for this limitation in the claim as it is not clear which “the site” is being referred to. Claim 1 also recites “a set of sites on the sequence read”.
Claims 18 and 21-22 are indefinite for depending from claim 17 or a dependent claim thereof and not rectifying the deficiency.
Regarding claim 21, the claim recites “wherein the second reference methylation pattern corresponds to other tissue types of the plurality of tissue types”. It is not clear whether “other tissue types” is intended to encompass “all other tissue types” of the plurality of tissue types, “another tissue type” of the plurality of tissue types, “one or more other tissue type” of the plurality of tissue types, or some other grouping. The instant specification recites “another tissue type” (para [0144]) and “one or more other tissue types” (para [0306], [0311], [0352], etc.) in the context of classifying.
Regarding claim 21 and 22, the claims recites “wherein the one of the plurality of tissue types [from which the sequence read is derived] includes…” either “the first tissue type” (claim 21) or “one of other tissue types” (claim 22). It is unclear how “the one” may “include” more than a single tissue type. That is to say, it is unclear whether “tissue type” is intended to have a different scope (i.e., be narrower) when referred to after an “ordinal” number adjective (e.g., “the one…tissue type” is liver tissue and “the first tissue type” is hepatocellular carcinoma). If this is the case, this is unclear and the choice of a different term (e.g., “tissue subtype”) is recommended. Alternately, this may be rectified by replacing “includes” with “is”.
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 1-24, 26, 41, and 64-65 are rejected under 35 U.S.C. 101 because the claimed invention is directed to a judicial exception without significantly more. The claim(s) recite(s) abstract ideas and natural phenomena. This judicial exception is not integrated into a practical application. The claim(s) does/do not include additional elements that are sufficient to amount to significantly more than the judicial exception.
The following three inquiries are used to determine whether a claim is drawn to patent-eligible subject matter:
Step 1. Is the claim directed to a process, machine, manufacture, or composition of matter?
Yes, claims 1-24, 26, 41, and 64-65 are directed to a process/method.
Step 2A, prong 1. Does the claim recite a law of nature, a natural phenomenon, or an abstract idea (recognized judicial exceptions)?
The claims are directed to analyzing a biological sample of a subject comprising receiving sequencing reads, comparing methylation patterns, determining a tissue classification of each sequence read, and determining a classification of a disease based on the tissue classifications of the sequence reads, as recited in claim 1. Receiving, comparing, and determining encompass the abstract ideas of mental processes including observation, evaluation, judgment, and forming an opinion—i.e., “receiving” may encompass observation/evaluation, “comparing” may encompass evaluation and judgement, and “determining” may encompass evaluation, judgement, and forming an opinion.
The claims also are directed to the abstract idea of mathematical calculations as the comparisons and classifications may encompass an algorithm that performs said comparisons and classifications using mathematical functions/models.
The claims likewise are directed to the natural phenomena of a methylation pattern of a sequence read being correlated with a tissue type and methylation patterns of sequence reads of tissue(s) of a sample being correlated with a disease in the organism from which the sample originates.
Step 2A, prong 2. Is the judicial exception(s) integrated into a practical application?
Regarding claim 1, as discussed above, each step encompasses only abstract ideas. While the sequence reads recite particular a class of sequencing, such not required step of the claim.
It is further noted for the purposes of compact prosecution that well-known steps of data gathering required for subsequent abstract steps would be considered insignificant extra-solution activity. See MPEP 2106.04(d)(I) and MPEP 2106.05(g).
Regarding 2-24, 26, 41, and 64-65, the additional limitations are directed to additional calculations that may encompass mental processes (i.e., abstract ideas), including machine learning classifications that encompass those may be conducted by hand (claims 5-6) or selecting a particular data source/type to be manipulated (e.g., types of disease, sequencing, etc.). Selecting data sources to be manipulated in the judicial exception is insignificant extra-judicial activity. See MPEP 2106.04(d)(I) and MPEP 2106.05(g)).
Step 2B. Does the claim amount to significantly more?
No, the claims fail to recite more than steps that encompass abstract ideas or a selection of a particular type of data to which to apply the abstract ideas (i.e., insignificant extra-solution activity).
As discussed in MPEP 2106.05(I), the inventive concept "cannot be furnished by the unpatentable law of nature (or natural phenomenon or abstract idea) itself." Genetic Techs. Ltd. v. Merial LLC, 818 F.3d 1369, 1376, 118 USPQ2d 1541, 1546 (Fed. Cir. 2016). See also Alice Corp., 573 U.S. at 21-18, 110 USPQ2d at 1981 (citing Mayo, 566 U.S. at 78, 101 USPQ2d at 1968 (after determining that a claim is directed to a judicial exception, "we then ask, ‘[w]hat else is there in the claims before us?") (emphasis added)); RecogniCorp, LLC v. Nintendo Co., 855 F.3d 1322, 1327, 122 USPQ2d 1377 (Fed. Cir. 2017) ("Adding one abstract idea (math) to another abstract idea (encoding and decoding) does not render the claim non-abstract").
Likewise, the courts have also found that insignificant extra-solution activity is not sufficient to qualify as “significantly more”. See MPEP 2106.05(A).
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claim(s) 1-10, 12, 14-22, 24, 26, 41, and 65 is/are rejected under 35 U.S.C. 102(a)(1) and (a)(2) as being anticipated by Zhou (WO 2017/212428 A1; published 12/14/2017; as cited in the IDS dated 05/31/2023).
Regarding claims 1, 8-10, and 14, Zhou teaches a method comprising:
receiving sequence reads obtained from a methylation-aware sequencing of a plurality of cfDNA molecules from a biological sample of a subject (para [00192] and Fig. 21, wherein the blood tube is interpreted to teach a biological sample; claim 1; see also claims 6, 11, and 15 and para [0009], [0025-26], [0094]);
for each of the sequence reads:
comparing a methylation pattern of said sequence read to a first reference methylation pattern corresponding to a tumor class [i.e., a first tissue type] of a plurality of classes (para [00192] and [00196-198]; Fig. 21; claim 2); and
based on the comparison between the methylation and the pattern and the first reference methylation pattern, determining a tissue classification of the sequence read, wherein the tissue is a tumor (para [00198]; Fig. 21; claim 2; instant claims 8-10; see also claims 7-8) and wherein the classification is based on a likelihood [i.e., probability] (para [00198]; Fig. 21; claim 2; instant claim 14); and
determining a tumor burden [i.e., a classification of a disease] in the sample based on the classification of the sequence reads (para [00199-200]; Fig. 21; claim 4, wherein the biological composition may be a cancer or diseased tissue, see claims 7-8)
See also claims 1, 2, 7-8, and 15-16; [0009-10], [0025-26], [0095-96], [00238-252].
Zhou teaches that the methylation sequencing data is obtained from nucleic acid sequence of 50 or more nucleic acids (claim 1); thus, the artisan would understand that the cfDNA would inherently have a “size range”. For the purposes of compact prosecution, Zhou further notes that a sequencing read may be a wide variety of sizes in para [00112], wherein the artisan would understand that the molecules sequenced would inherently be at least as large as the continuous read length.
Alternatively/additionally, Zhou teaches integrating the information into a disease diagnosis [i.e., classification of a disease] after the composition and tissue origin of the cfDNA are determined (para [00121]) based on the classification of the sequence reads (para [00122]).
Regarding claim 2, Zhou teaches that determining the level of the of tumor burden/biological composition is based on the number of sequencing reads that are tumor-derived/containing the biological composition (para [00199-200]; Fig. 21; claim 4), and that that the diagnosis method is also based on a first amount of sequence reads classified as being the tumor (para [00226-230] and para [00122]).
Regarding claim 3-4 and 7, Zhou teaches a Z-score based integration method for binary and multi-class classification (para [00228-229]; Fig. 7 and 9; instant claim 7), wherein a Z-score compares the amount of reads to the reference type of known classification (para [00122]; instant claims 3-4) and wherein such is used for diagnosis (para [00121]). Zhou teaches comparing the “score” to that of normal people (para [00233]).
For claim 4, “a reference sample” is interpreted broadly to mean one or more reference samples in view of instant para [0435] which states that “a” is intended to mean “one or more” absent an indication otherwise. Thus, a cutoff based on a mean of reference samples fulfills the limitation.
Regarding claims 5-6, Zhou teaches that the methods comprise applying a machine-learning model, broadly interpreted, to predict a probability based on the frequencies [i.e., amounts] of the reads, indicative of the classification of the disease (para [00205-215]; Fig. 11; para [00228-00231]; see also claim 142; instant claim 5), wherein the training samples were of known classification (para [00227], [00251], [00255], [001153-154]; instant claim 6).
Regarding claim 7, Zhou also teaches inferring compositions of multiple tissue types based on the methylation patterns of individual reads and determining (para [00205-2016]) and using that in a multi-class diagnosis (para [00228-229]; Fig. 9; see also para [00121]).
Regarding claim 12, Zhou teaches calculating the methylation pattern based on the sequencing read, wherein the methylation pattern comprises a genomic region [i.e., location] corresponding to the nucleic acid sequence [of said read] and the methylation statuses in the genomic region (claims 1 and 10; para [00196]).
Zhou also teaches that the methods provide tumor location/tissue-of-origin information (para [00127]), such that the artisan would understand that a “location” of the sequence read and a methylation pattern would correspond to the tissue-of-origin of said read and the corresponding methylation pattern by which it’s identified (see, e.g., Fig. 4 and Fig. 6).
Regarding claims 15-16, Zhou teaches that the larger the prediction score (interpreted to be the Z-score used in the classification of the disease; see Fig. 10), the later the cancer stage (para [00232]).
Regarding claims 17-20 and 24, Zhou teaches averaging (instant claim 20) the methylation values of all CpG sites in a given read and determining the difference between the “normal” and “tumor” cfDNAs (para [00190]; instant claim 24; see also para [00142]). The “normal” cfDNAs are scored as a percentage [i.e., a methylation index; instant claim 17] (para [00191]).
The ordinarily-skilled artisan would understand that subtracting values of each set of sites before taking an average [i.e., aggregate value] would be mathematically equivalent to the taught average and determining a difference [i.e., similarity metric] between the values, i.e., mean (a – b) = mean (a) – mean (b) (instant claim 18).
Zhou teaches that the methylation values may be represented as binary values in the calculation (para [00190]; see also, e.g., para [00197]; instant claim 19).
Zhou also teaches calculating the likelihood of the joint methylation status in read r for methylation pattern m P(r|m) where metric is calculated as a product of the conditional probabilities for each CpG site j [i.e., similarity metric] (instant claim 18), wherein the joint methylation status is represented as a binary value (instant claim 19), wherein such is implemented for particular tissue classes (para [00198]).
Regarding claims 21 and 22, Zhou teaches determining the likelihood values for the read based on the aggregate of each site (Fig. 22). Zhou teaches maximizing the likelihood (para [00199]), i.e., choosing the tumor class when the aggregate of the tumor pattern is greater than that of the normal pattern or choosing the normal class when the aggregate of the tumor pattern is less than that of the normal pattern (instant claims 21-22).
Regarding claim 26, Zhou teaches the cancer may be hepatocarcinoma [interpreted as hepatocellular carcinoma; see col 76, para 5], lung, breast, glioblastoma, pancreatic, colon, head, neck, gum, or tongue cancer.
Regarding claims 41 and 65, Zhou teaches that the sequencing reads are whole genome bisulfite or reduced representation bisulfite (instant claim 65) covering at least 3 CpG sites (instant claim 41; see also para [00250]) (para [0089]).
For at least these reasons, claims 1-10, 12, 14-22, 24, 26, 41, and 65 are anticipated by Zhou.
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 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.
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.
Claim(s) 1-10, 12-24, 26, 41, and 65 is/are rejected under 35 U.S.C. 103 as being unpatentable over Zhou (WO 2017/212428 A1; published 12/14/2017; as cited in the IDS dated 05/31/2023).
Regarding claims 1, 8-10, and 14, Zhou teaches a method comprising:
receiving sequence reads obtained from a methylation-aware sequencing of a plurality of cfDNA molecules from a biological sample of a subject (para [00192] and Fig. 21, wherein the blood tube is interpreted to teach a biological sample; claim 1; see also claims 6, 11, and 15 and para [0009], [0025-26], [0094]);
for each of the sequence reads:
comparing a methylation pattern of said sequence read to a first reference methylation pattern corresponding to a tumor class [i.e., a first tissue type] of a plurality of classes (para [00192] and [00196-198]; Fig. 21; claim 2); and
based on the comparison between the methylation and the pattern and the first reference methylation pattern, determining a tissue classification of the sequence read, wherein the tissue is a tumor (para [00198]; Fig. 21; claim 2; instant claims 8-10; see also claims 7-8) and wherein the classification is based on a likelihood [i.e., probability] (para [00198]; Fig. 21; claim 2; instant claim 14); and
determining a tumor burden [i.e., a classification of a disease] in the sample based on the classification of the sequence reads (para [00199-200]; Fig. 21; claim 4, wherein the biological composition may be a cancer or diseased tissue, see claims 7-8)
See also claims 1, 2, 7-8, and 15-16; [0009-10], [0025-26], [0095-96], [00238-252].
Zhou teaches that the methylation sequencing data is obtained from nucleic acid sequence of 50 or more nucleic acids (claim 1); thus, the artisan would understand that the cfDNA would inherently have a “size range”. For the purposes of compact prosecution, Zhou further notes that a sequencing read may be a wide variety of sizes in para [00112], wherein the artisan would understand that the molecules sequenced would inherently be at least as large as the continuous read length.
Alternatively/additionally, Zhou teaches integrating the information into a disease diagnosis [i.e., classification of a disease] after the composition and tissue origin of the cfDNA are determined (para [00121]) based on the classification of the sequence reads (para [00122]).
Regarding claim 2, in the method of Zhou, Zhou teaches that determining the level of the of tumor burden/biological composition is based on the number of sequencing reads that are tumor-derived/containing the biological composition (para [00199-200]; Fig. 21; claim 4), and that that the diagnosis method is also based on a first amount of sequence reads classified as being the tumor (para [00226-230] and para [00122]).
Regarding claim 3-4 and 7, in the method of Zhou, Zhou teaches a Z-score based integration method for binary and multi-class classification (para [00228-229]; Fig. 7 and 9; instant claim 7), wherein a Z-score compares the amount of reads to the reference type of known classification (para [00122]; instant claims 3-4) and wherein such is used for diagnosis (para [00121]). Zhou teaches comparing the “score” to that of normal people (para [00233]).
For claim 4, “a reference sample” is interpreted broadly to mean one or more reference samples in view of instant para [0435] which states that “a” is intended to mean “one or more” absent an indication otherwise. Thus, a cutoff based on a mean of reference samples fulfills the limitation.
Regarding claims 5-6, in the method of Zhou, Zhou teaches that the methods comprise applying a machine-learning model, broadly interpreted, to predict a probability based on the frequencies [i.e., amounts] of the reads, indicative of the classification of the disease (para [00205-215]; Fig. 11; para [00228-00231]; see also claim 142; instant claim 5), wherein the training samples were of known classification (para [00227], [00251], [00255], [001153-154]; instant claim 6).
Regarding claim 7, in the method of Zhou, Zhou also teaches inferring compositions of multiple tissue types based on the methylation patterns of individual reads and determining (para [00205-2016]) and using that in a multi-class diagnosis (para [00228-229]; Fig. 9; see also para [00121]).
Regarding claim 12, in the method of Zhou, Zhou teaches calculating the methylation pattern based on the sequencing read, wherein the methylation pattern comprises a genomic region [i.e., location] corresponding to the nucleic acid sequence [of said read] and the methylation statuses in the genomic region (claims 1 and 10; para [00196]).
Zhou also teaches that the methods provide tumor location/tissue-of-origin information (para [00127]), such that the artisan would understand that a “location” of the sequence read and a methylation pattern would correspond to the tissue-of-origin of said read and the corresponding methylation pattern by which it’s identified (see, e.g., Fig. 4 and Fig. 6).
Regarding claims 15-16, in the method of Zhou, Zhou teaches that the larger the prediction score (interpreted to be the Z-score used in the classification of the disease; see Fig. 10), the later the cancer stage (para [00232]).
Regarding claims 17-20 and 24, in the method of Zhou, Zhou teaches averaging (instant claim 20) the methylation values of all CpG sites in a given read and determining the difference between the “normal” and “tumor” cfDNAs (para [00190]; instant claim 24; see also para [00142]). The “normal” cfDNAs are scored as a percentage [i.e., a methylation index; instant claim 17] (para [00191]).
The ordinarily-skilled artisan would understand that subtracting values of each set of sites before taking an average [i.e., aggregate value] would be mathematically equivalent to the taught average and determining a difference [i.e., similarity metric] between the values, i.e., mean (a – b) = mean (a) – mean (b) (instant claim 18).
Zhou teaches that the methylation values may be represented as binary values in the calculation (para [00190]; see also, e.g., para [00197]; instant claim 19).
Zhou also teaches calculating the likelihood of the joint methylation status in read r for methylation pattern m P(r|m) where metric is calculated as a product of the conditional probabilities for each CpG site j [i.e., similarity metric] (instant claim 18), wherein the joint methylation status is represented as a binary value (instant claim 19), wherein such is implemented for particular tissue classes (para [00198]).
Regarding claims 21 and 22, in the method of Zhou, Zhou teaches determining the likelihood values for the read based on the aggregate of each site (Fig. 22). Zhou teaches maximizing the likelihood (para [00199]), i.e., choosing the tumor class when the aggregate of the tumor pattern is greater than that of the normal pattern or choosing the normal class when the aggregate of the tumor pattern is less than that of the normal pattern (instant claims 21-22).
Regarding claim 26, in the method of Zhou, Zhou teaches the cancer may be hepatocarcinoma [interpreted as hepatocellular carcinoma; see col 76, para 5], lung, breast, glioblastoma, pancreatic, colon, head, neck, gum, or tongue cancer.
Regarding claims 41 and 65, in the method of Zhou, Zhou teaches that the sequencing reads are whole genome bisulfite or reduced representation bisulfite (instant claim 65) covering at least 3 CpG sites (instant claim 41; see also para [00250]) (para [0089]).
Regarding claims 13 and 23, in the method of Zhou, Zhou fails to explicitly teach that the methylation pattern includes a number of bases between pairs of sites of the set of sites in the read-based embodiments (instant claim 13) and classifying the disease based on a “highest amount” of sequencing reads associated with a “first tissue type” (instant claim 23).
Zhou rectifies this by teaching that individual CpGs may be grouped into CpG clusters in order to use more mappable reads, wherein two adjacent CpG sites are grouped into one CpG cluster if their flanking regions (e.g., of 100 bp) overlap (para [00134]), thereby requiring a minimum number of bases between pairs of sites of the set of sites of a methylation pattern (instant claim 13).
Zhou teaches that the flanking regions may be a range between 25 to 300 and merging clusters if they overlap (e.g., claims 129 and 143).
Zhou teaches a relationship between numbers of predictions and predicted ctDNA burden wherein the higher the predicted circulating tumor DNA burden, the higher percentage of the predicted number of samples originated from individuals with cancer (Fig. 14A; instant claim 23).
Zhou teaches optimizing the method based on higher estimated tumor burdens and a patient with a majority 59% tumor burden that was correctly classified (para [00155]; Fig. 14B; Fig. 15).
Therefore, it would have been obvious to one ordinary skill in the art before the effective filing date of the claimed invention to include the number of bases between pairs of sites of the set of sites as a feature of the pattern, motivated by the desire to use more mappable reads, as taught by Zhou, and to ensure biologically distinct/meaningful differentially methylated sites. There would have been a strong expectation of success as both methods are directed toward measurement of methylation of cfDNA samples and such represents the application of a known technique to a known method.
Further, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to substitute and/or further include the highest amount of sequence read as part of the classification as part of the routine optimization, motivated by a desire to reduce possible false positives, as taught by Zhou. Such would have had a high degree of success as such a modification of the algorithm(s) would have been within the skillset of the artisan and Zhou teaches various methods of setting thresholds and considering amount of reads.
Claim(s) 11 is/are rejected under 35 U.S.C. 103 as being unpatentable over Zhou (WO 2017/212428 A1; published 12/14/2017; as cited in the IDS dated 05/31/2023) as applied to claim 10 above, and in view of Liao (Liao W, et al. Noninvasive detection of tumor-associated mutations from circulating cell-free DNA in hepatocellular carcinoma patients by targeted deep sequencing. Oncotarget. 2016 Jun 28;7(26):40481-40490).
Regarding claim 11, in the method of Zhou, Zhou teaches that organs with strong blood circulation can result in high tumor burden (para [00155]), utilizing tumors in patents with and without large blood flow (para [00160]), and that the cancer may be metastatic and any stage (para [0035]), but fails to explicitly teach determining whether vascular invasion exists.
Liao rectifies this by teaching a method of detecting vascular invasion in cfDNA using sequencing, wherein the vascular invasion associated mutations predicted shorter recurrence-free survival time (Abstract).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have further determined whether vascular invasion exists, motivated by the desire to better predict recurrence-free survival time, as taught by Liao. There would have been a strong expectation of success as both utilize sequencing-based methods applied to cfDNA of known or possible cancer patients to perform a classification, wherein such represents the application of a known technique to a known method.
Claim(s) 64 is/are rejected under 35 U.S.C. 103 as being unpatentable over Zhou (WO 2017/212428 A1; published 12/14/2017; as cited in the IDS dated 05/31/2023) as applied to claim 1 above, and in view of Liu (Liu Y, et al. DNA methylation-calling tools for Oxford Nanopore sequencing: a survey and human epigenome-wide evaluation. Genome Biol. 2021 Oct 18;22(1):295).
Regarding claim 64, Zhou fails to teach that the methylation-aware sequencing does not include bisulfite sequencing.
Liu rectifies this by teaching nanopore long-read sequencing for per-read and per-site evaluation of CpG methylation (Abstract). Liu teaches nanopore sequencing directly detects DNA, enabling rapid long-read sequencing, and single-based, single-molecule sensitivity (Background, para 3), including directly calling methylation modifications from sequencing signals (e.g., Background, para 4-5).
Liu teaches that bisulfite conversion damages DNA (Background, para 1) and that it cannot distinguish between 5mCs and 5hmCs, unlike nanopore sequencing (Conclusion, para 2).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to simply substitute the methylation sequencing of Zhou with the nanopore sequencing of Liu, motivated by the desire to avoid damage of the DNA and to increase relevant information by being able to distinguish between 5mC and 5hmC, as taught by Liu. There would have been a strong expectation of success as both are directed to methylation analysis, including of per molecule and per site calls. Further, such represents a substitution of equivalents known for the same purpose, i.e., methylation aware sequencing. See MPEP 2144.06.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
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Claims 1-10, 12-24, 41, and 65 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-38 of U.S. Patent No. 12,139,750 B2 in view of Zhou (WO 2017/212428 A1; published 12/14/2017; as cited in the IDS dated 05/31/2023), Liao (Liao W, et al. Noninvasive detection of tumor-associated mutations from circulating cell-free DNA in hepatocellular carcinoma patients by targeted deep sequencing. Oncotarget. 2016 Jun 28;7(26):40481-40490), and Liu (Liu Y, et al. DNA methylation-calling tools for Oxford Nanopore sequencing: a survey and human epigenome-wide evaluation. Genome Biol. 2021 Oct 18;22(1):295), evidenced MedlinePlus (Choriocarcinoma: Medlineplus medical encyclopedia [Internet]. U.S. National Library of Medicine; [cited 2026 Jan 17]. Available from: https://medlineplus.gov/ency/article/001496.htm).
Both sets of claims are encompass methods of receiving sequencing reads with a size range, and, for each read/cfDNA to which a read would correspond, determining a methylation status at each site of a plurality of sites, determining a methylation pattern using the methylation statuses, comparing the methylation pattern to one or more reference patterns determined for a particular tissue type, and determining a tissue of origin, of the cfDNA molecule using the comparison (claim 1), wherein the sequence read may be identified as a haplotype present in the female and determined to be a fetal tissue of origin using the methylation pattern, wherein the haplotype is further identified as carrying a disease-causing genetic mutation or variation and the fetus is classified as likely to have the disease caused by the genetic mutation or variant (claims 15).
It is noted that determining per-read versus per-cfDNA would be a matter of routine optimization, in part, based on sequencing depth as low sequencing depth assays would be statistically unlikely to have multiple unique reads per locus. Further, interpreted broadly, methods may include molecular identifiers and means of collapsing “read” information to perform error correction such that a “read” may be comprised of multiple sequencing passes.
‘750 teaches determining an amount and determining the fractional contribution of the tissue of origin (claim 5), i.e., a basis of the tissue of origin classification; determining the tissue of origin based on a machine learning model (claims 17-18), a genomic location of the reads (claim 15); utilizing the distance between sites [i.e., bases between pairs] (claim 20); a probability and similarity score (claim 25); aggregate value that is the product of the plurality of probabilities (claim 26); and that the plurality of sites comprises at least 5 CpG sites (claim 22).
‘750 fails to explicitly teach all of the comparisons, severity of disease, the methylation index and binary value representations, all aggregate values and comparisons, all classifications, that one tissue is without disease, what the methylation aware sequencing is, vascular invasion, that the disease is cancer, and particular types of cancer.
As discussed in the 103 rejection above, Zhou teaches and/or suggests that: the comparison of the comparison of the first amount may be compared to a cutoff value and based on a reference sample of known classification; severity of disease; methylation index and binary values representing methylation status; additional aggregate values and comparisons thereof; additional classifications based on comparisons; explicitly that one tissue may be without disease; sequencing with bisulfite; and classifying for other diseases and cancer types.
Zhou teaches that joint methylation patterns of multiple adjacent CpG sites on an individual cfDNA sequencing read allow for signal amplification and allow for more sensitive capture of abnormal methylation signal affecting smaller proportions of cfDNAs (para [00190]), especially as aberrant DNA methylation patterns occur early in the pathogenesis of diseases including cancer, facilitating early detection (para [00189]). Zhou teaches that its approach outperformed previous methods that relied on averages in both prediction performance and robustness and greater consistency with clinical information, even with low sequencing coverage (para [00191]).
As discussed in the 103 rejection above, Liao teaches vascular invasion, wherein the vascular invasion associated mutations predicted shorter recurrence-free survival time (Abstract).
As discussed in the 103 rejection above, Liu teaches a method of non-bisulfite sequencing and that that bisulfite conversion damages DNA (Background, para 1) and that it cannot distinguish between 5mCs and 5hmCs, unlike nanopore sequencing (Conclusion, para 2).
Regarding claim 10, as evidenced by MedlinePlus, choriocarcinoma are cancers that can develop at the beginning of a pregnancy from cells that normally become the placenta [i.e., the trophoblast, which is of fetal genetic origin]. As such, it would be expected to meet the limitation of a fetal disease caused by disease-causing genetic mutation/variation, which may be of maternal origin, and a cancer. MedlinePlus teaches the cancer most often occurs with a complete hydatidiform mole, i.e., the fetus (broadly interpreted) is affected by [i.e., “has”] the same diseased caused by genetic mutation/variant.
Any additional limitations of the ‘750 claims are encompassed by the open claim language “comprising” found in the instant claims.
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have combined ‘750 with the methods of Zhou, motivated by the desire to improve prediction performance and robustness, even with low sequencing coverage, as taught by Zhou. There would have been a strong expectation of success, as both are directed to cfDNA methods utilizing methylation markers, and such represents the application of known techniques to known methods.
Further, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have further determined whether vascular invasion exists in the combined method of ‘750 and Zhou, motivated by the desire to better predict recurrence-free survival time, as taught by Liao. There would have been a high expectation of success as all utilize sequencing-based methods applied to cfDNA of known or possible cancer patients to perform a classification, wherein such represents the application of a known technique to a known method.
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to simply substitute the methylation sequencing in the combined method with the nanopore sequencing of Liu, motivated by the desire to avoid damage of the DNA and to increase relevant information by being able to distinguish between 5mC and 5hmC, as taught by Liu. There would have been a high probability of success as both are directed to methylation analysis, including of per molecule and per site calls. Further, such represents a substitution of equivalents known for the same purpose, i.e., methylation aware sequencing. See MPEP 2144.06.
Claims 1-10, 12-24, 26, 41, and 65 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-38 of U.S. Patent No. 10,392,666 B2 in view of Zhou (WO 2017/212428 A1; published 12/14/2017; as cited in the IDS dated 05/31/2023) and Liao (Liao W, et al. Noninvasive detection of tumor-associated mutations from circulating cell-free DNA in hepatocellular carcinoma patients by targeted deep sequencing. Oncotarget. 2016 Jun 28;7(26):40481-40490).
Both sets of claims are directed to analyzing a plurality of sequence reads from methylation-aware sequencing of a plurality of cfDNA molecules in a biological sample, wherein the sequencing includes one or more methylation sites and the status of each site is determined and a determining a classification level of cancer based on a comparison of the respective number of cfDNA molecules methylated (claim 1).
’666 also recites that the cfDNA molecules have a size range (e.g., claims 45-49); the methylation-aware sequencing may (claims 2-3) or may not (claim 5) use bisulfite treatment; determining a fractional concentration of tumor DNA and calculating a cutoff based on that amount (claim 42); calculating a sum of a cumulative score of CpG sites in a CpG island (claim 57)
The claims of ‘666 differ from the instant claims by reciting that the methylation level is calculated based on a number of cfDNA molecules methylated at each of the plurality of sites [wherein a plurality may be interpreted to be a “pattern”], rather than comparing each sequencing read to the reference pattern/level.
‘666 also fails to explicitly teach: determining the fractional contribution of the tissue of origin; determining the tissue of origin based on a machine learning model; a genomic location of the reads; utilizing the distance between sites [i.e., bases between pairs]; a similarity score for a given read; the aggregate value using a methylation index; the plurality of sites comprises at least 3 CpG sites; severity of disease; the methylation index and binary value representations; all of the comparisons; vascular invasion; and particular types of cancer.
As discussed in the 103 rejection above, Zhou teaching per-read comparisons of a joint plurality of sites on a sequencing read from a cfDNA molecule [i.e., a methylation pattern]; classification of the read as originating from a “tissue” based on said methylation pattern; and determining the classification based on the tissue classifications.
Zhou teaches that joint methylation patterns of multiple adjacent CpG sites on an individual cfDNA sequencing read allow for signal amplification and allow for more sensitive capture of abnormal methylation signal affecting smaller proportions of cfDNAs (para [00190]), especially as aberrant DNA methylation patterns occur early in the pathogenesis of diseases including cancer, facilitating early detection (para [00189]). Zhou teaches that its approach outperformed previous methods that relied on averages in both prediction performance and robustness and greater consistency with clinical information, even with low sequencing coverage (para [00191]).
As discussed in the 103 rejection above, Zhou also teaches and/or suggests each of determining the fractional contribution of the tissue of origin; determining the tissue of origin based on a machine learning model; utilizing genomic location of the reads; utilizing the distance between sites [i.e., bases between pairs]; utilizing the similarity score for a given read; utilizing the aggregate value using a methylation index; that a plurality of sites on a cfDNA comprises at least 3 CpG sites; determining a severity of disease; utilizing the methylation index and binary value representations; each of the comparisons; and particular types of cancer.
As discussed in the 103 rejection above, Zhou teaches and/or suggests that: the comparison of the comparison of the first amount may be compared to a cutoff value and based on a reference sample of known classification; severity of disease; methylation index and binary values representing methylation status; additional aggregate values and comparisons thereof; additional classifications based on comparisons; explicitly that one tissue may be without disease; sequencing with bisulfite; and classifying for other diseases and cancer types.
Zhou teaches that joint methylation patterns of multiple adjacent CpG sites on an individual cfDNA sequencing read allow for signal amplification and allow for more sensitive capture of abnormal methylation signal affecting smaller proportions of cfDNAs (para [00190]), especially as aberrant DNA methylation patterns occur early in the pathogenesis of diseases including cancer, facilitating early detection (para [00189]). Zhou teaches that its approach outperformed previous methods that relied on averages in both prediction performance and robustness and greater consistency with clinical information, even with low sequencing coverage (para [00191]).
As discussed in the 103 rejection above, Liao teaches vascular invasion, wherein the vascular invasion associated mutations predicted shorter recurrence-free survival time (Abstract).
Any additional limitations of the ‘750 claims are encompassed by the open claim language “comprising” found in the instant claims.
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have combined ‘750 with the methods of Zhou, motivated by the desire to improve prediction performance and robustness, even with low sequencing coverage, as taught by Zhou. There would have been a strong expectation of success, as both are directed to cfDNA methods utilizing methylation markers, and such represents the application of known techniques to known methods.
Further, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have further determined whether vascular invasion exists in the combined method of ‘750 and Zhou, motivated by the desire to better predict recurrence-free survival time, as taught by Liao. There would have been a high expectation of success as all utilize sequencing-based methods applied to cfDNA of known or possible cancer patients to perform a classification, wherein such represents the application of a known technique to a known method.
Conclusion
No claims are allowed.
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/EMMA R HOPPE/Examiner, Art Unit 1683
/NANCY J LEITH/Primary Examiner, Art Unit 1636