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 .
Election/Restrictions
Applicant’s election without traverse of Group II (claims 30 and 44) in the reply filed on 7/23/2026 is acknowledged.
Claims 2-29 have been canceled. Claims 74-101 have been added and depend on claim 30. Claims 1, 61, and 68 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to nonelected inventions, there being no allowable generic or linking claim. It is noted that Applicant has not provided the correct status identifiers for the withdrawn claims (i.e. (Withdrawn)). Applicant should add these upon their subsequent response in accordance with MPEP 1893.01(a)(4).
Claims 30, 44, and 74-101 are pending and are examined on the merits herein
Information Disclosure Statement
The information disclosure statements (IDS) submitted on 5/7/2024, 7/22/2024, and 7/23/2026 are in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
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: reference characters 465 and 470 in Figure 13. 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.
Claim Objections
Claim 83 is objected to because of the following informality: in line 1, “converted cfDNA molecules” should read “converted cfDNA fragments” to better match the language used in claim 30, from which this claim depends. Appropriate correction is required.
Claim 88 is objected to because of the following informality: in line 2, “a one or more preservative reagent” should read “one or more preservative reagents.” Appropriate correction is required.
Claim Rejections - 35 USC § 112(a)
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 30, 44, and 74-101 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for examining bladder cancer, prostate cancer, and kidney cancer with the genes in Tables 2-3, 4, and 5 respectively, does not reasonably provide enablement for examining all cancers with all of the genes present in instant Table 1. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention commensurate in scope with these claims.
Factors to be considered in determining whether a disclosure meets the enablement requirement of 35 USC 112, first paragraph, have been described by the court in In re Wands, 8 USPQ2d 1400 (CA FC 1988). These factors include (1) the quantity of experimentation necessary, (2) the amount of direction or guidance presented, (3) the presence or absence of working examples, (4) the nature of the invention, (5) the state of the prior art, (6) the relative skill of those in the art, (7) the predictability or unpredictability of the art, and (8) the breadth of the claims. Each of these factors is discussed below.
Nature of the Invention
Claims 30, 44, and 74-101 are drawn to a method for detecting cancer cells in a urine sample of a subject with cfDNA analysis, sequencing, and the use of a trained classifier.
The claimed methods are classified in the unpredictable arts of molecular biology and biochemistry.
Breadth of the Claims
Claim 30 is very broad in scope because it encompasses any type of cancer, and a wide variety of genes as presented in Table 1.
The only dependent claim that limits the type of cancer presented in claim 30 is claim 83, which requires the cancer cells to be one of bladder cancer, prostate cancer, or kidney cancer. This claim also limits the genes that may be analyzed in association with each cancer.
Claim 99 also recites that cancer cells may be bladder cancer, prostate cancer, or kidney cancer cells, but it is noted that this claim states that the cancer cells may comprise these types of cells, and so other cell types are not excluded. This claim also does not limit the genes that may be analyzed with each cell type.
Level of Skill in the Art
The ordinary artisan typically holds at least a master’s degree has several years of experience.
State of the Prior Art & Unpredictability
Concerning the claimed invention, what was (and is) unpredictable in the art is whether a particular biomarker listed in Table 1 of the instant specification would be useful in the context of instant claim 30 to detect any type of cancer. Different biomarkers may have different sensitivities and may be more or less useful in different contexts, and so may not be informative in the context of a classifier for a particular cancer.
For instance, in Larsen et al. (Int. J. Mol. Sci., 2019), which discusses DNA methylation biomarkers for various urological cancers, HOXD3, RASSF1, and CDH1 have sensitivities above 60% for prostate cancer (Figure 2), while for bladder cancer, HOXD3 is not examined, and CDH1 and RASSF1 have much lower sensitivities. For renal cancer, HOXD3 is also not listed as a biomarker, CDH1 has a sensitivity of 38% on its own, and RASSF1A has a sensitivity of 65% when examined on its own (Table 4). Thus, for any given cancer, both the choice of biomarker and the accuracy of a biomarker can vary.
Ellinger et al. (Expert Review of Molecular Diagnostics, 2015) discusses biomarkers for bladder, prostate, renal, and testicular cancer (Abstract). The various tables of the invention denote DNA methylation biomarkers for each cancer. Table 1 notes that GSTP1 is a main biomarker for prostate cancer, while this is not a biomarker for DNA methylation in bladder and kidney cancer (Tables 2-3). While bladder and kidney cancer do share some DNA methylation biomarkers (e.g. CDH1), there are also many biomarkers that they do not share (e.g. PCDH10 in bladder cancer in Table 2). A similar pattern is shown when comparing DNA methylation markers for testicular cancer (Table 4) and the other cancers, where some biomarkers are in common but others are unique.
Locke et al. (Frontiers in Genetics, 2019) discusses various DNA methylation biomarkers for various cancers, and Table 1 in particular lists current biopsy tests for various cancers and the biomarkers associated with them, where the biomarker panels differ for different cancers (e.g. SEPT9 is used in hepatocellular carcinoma and colorectal cancer but not bladder cancer).
Thus, it is known in the art that different cancers will have different profiles of informative biomarkers, and for a given set of biomarkers, it is not immediately clear how informative the set will be for a particular cancer.
Guidance in the Specification and Examples
Regarding the types of cancers analyzed, the instant specification does discuss cancer generally (e.g. para. 7). Bladder cancer, prostate cancer, and kidney cancer are specifically recited throughout (e.g. paras. 11, 19, and 102) The specification does mention the more broad term of urological cancers (e.g. paras. 98 and 119), but this appears more as a means to characterize the bladder, prostate, and kidney cancers. No other cancers are specifically mentioned. It is noted that liver and breast cancer are used as examples that may be represented in a probability function in para. 249, but the actual use of liver and/or breast cancer is not recited.
Regarding the genes analyzed, paras. 11, 15, and 21 (among others) mention TWIST1, EOMES, HOXA9, POU4F2, and ZNF154, which are the genes described in instant claim 81. Tables 1-5, which provide gene tables, are not directly noted to be associated with particular cancers outside of their disclosure in the working examples (though see below). It is noted that TWIST1 appears in Tables 1-3, but none of EOMES, HOXA9, POU4F2, and ZNF154 appear in any of the tables.
In the working examples of the instant specification, Example 2 analyzed bladder, prostate, and kidney cancer in urine samples (paras. 295-296). Para. 300 states, “Genomic regions of the genes in Tables 2 and 3 were found to contain methylation markers of bladder cancer. Genomic regions of the genes in Table 4 were found to contain methylation markers of prostate cancer. Genomic regions of the genes in Table 5 were found to contain methylation markers of kidney cancer. Table 1 presents the union of genes in Tables 2-5.”
Thus, Applicant’s specification does not appreciably cover any cancers other than bladder, kidney, or prostate cancer, and the working examples specifically note that particular methylation biomarkers are associated with particular cancers. These biomarker determinations were based on whole genome sequences (see para. 296), and so are not the result of only choosing a subset of genes to examine.
In view of the unpredictability in the art discussed above, it is not clear that the results discussed by Applicant would extend over the full scope of the claimed invention.
Quantity of Experimentation
The ordinary artisan would have to conduct a very large quantity of highly unpredictable experimentation before being able to successfully practice the full scope of the claimed methods. Specifically, the ordinary artisan would have to determine that each biomarker in Table 1 is capable of functioning as a biomarker for every type of cancer encompassed by the claims. Based on the teachings in the art, this would be an inventive and unpredictable undertaking, requiring extensive experimentation in which there is no guarantee of success. The large quantity of experimentation and its unpredictability constitute undue experimentation.
Conclusion
In view of the foregoing, it is clear that the specification fails to enable the full scope of the claimed methods, and claims 30, 44, and 74-101 are rejected under 35 U.S.C. 112(a) for failing to comply with the enablement requirement.
With regard to what is considered enabled, bladder cancer cells are considered to be detectable using the genes described in Tables 2-3 of the instant specification, prostate cancer cells are considered to be detectable using the genes described in Table 4 of the instant specification, and kidney cancer cells are considered to be detectable using the genes described in Table 5 of the instant specification.
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.
Claims 30, 44, and 74-101 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 30, MPEP 2173.05(s) states, “Where possible, claims are to be complete in themselves…Reference characters corresponding to elements recited in the detailed description and the drawings may be used in conjunction with the recitation of the same element or group of elements in the claims.” Claim 30 recites Table 1 of the specification without also reciting the elements within the table, and so this claim is indefinite. As well, there is no particular reason why the information in the table cannot be presented in the claim.
Claims 44 and 74-101 are rejected based on their dependence on rejected claim 30.
Similarly, claim 80 is also rejected for reciting Table 1 without also reciting the elements within the table. Claim 82 is also similarly rejected for reciting Tables 2-5 without also reciting the elements within the tables.
Claim 79 is also rejected for reciting “the application of the trained classifier” in line 1, as “an application of the trained classifier” is not recited earlier in the claim or in claims 30, 76, or 78, from which this claim depends, and so this phrase lacks antecedent basis. This is further compounded by the use of “further comprises” in line 2 of the claim, as “the application” is not stated to comprise any elements before this point. It will be interpreted as though this application is referring to the use of the trained classifier in step (d) of claim 30. It is recommended to amend this claim to clearly link the language to step (d) of claim 30 if that is Applicant’s intention.
Claim 94 is also rejected because it generally recites the increase of “a concentration,” but does not specify what component of the retained urine sample has an increased concentration. It will be interpreted as though this is referring to the concentration of cfDNA in the retained urine sample described in claim 44, from which this claim depends. It is recommended that Applicant amend the claim to read “wherein the retained urine sample has a concentration of cfDNA fragments that is increased by at least…compared to that of the purified urine sample.”
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 30, 44, and 74-100 are rejected under 35 U.S.C. 101 because the claimed invention is directed to a judicial exception without significantly more. The claims recite a natural law.
Claim 30 is directed to a method for identifying cancer cells within the urine sample of a subject based on the expression of particular genes. The natural law recited is the correlation of the gene expression values and the presence of cancer in a sample. This judicial exception is not integrated into a practical application because there is no required active treatment step or other step that integrates the judicial exception into a practical application. See MPEP 2106.04(d). The claim does not include additional elements that are sufficient to amount to significantly more than the judicial exception because they do not amount to more than well-understood, routine, and conventional activity in view of Salhia et al. (WO 2022/178108 A1).
Salhia teaches that methods of their invention can be used to determine the methylation level of genomic regions implicated in bladder carcinomas (pages 59-60, joining para.). Page 9, para. 4 also notes that bladder cancer is included in the general term cancer. Table 1 shows target genomic regions, including regions in the genes CRTC1 (page 53) and KNDC1 (page 41), which appear in instant Tables 1-3. These genes can specifically be used to determine the presence of cancer cells present in a patient (page 21, para. 4-5). A biological sample of the invention can be cell-free genomic DNA from a urine sample (page 9, para. 2).
Salhia is primarily drawn to methods related to ovarian cancer (e.g. Abstract). Regarding ovarian cancer, on page 16, para. 4, the reference teaches that the presence of ovarian cancer can be gleaned by using hybrid capture probes, such as pull-down probes/bait sets, to selectively enrich specific DNA molecules. These probes can correlate to the genes described in Table 1, and can specifically hybridize to one or more genomic regions in a cell-free DNA sample, and specifically to differentially methylated regions, such as CpG sites, The probes can be nucleic acid molecules (page 17, para. 1).
In Example 1 of the reference, a method is described where cell-free DNA from the plasma of ovarian cancer patients and normal controls was bisulfite converted, amplified, and sequenced, and this data was then used to construct a novel classifier that used machine learning to differentiate between ovarian cancer and benign tumors (see page 68). Differentially methylated regions were identified and biotinylated RNA probes were developed, where each probe was 60-80 nucleotides in length (page 69, para. 3. Then, cfDNA from plasma samples of a new cohort of patients with benign and malignant masses were taken. DNA libraries were created, bisulfite converted, and enriched with the previously created biotinylated probes used for hybrid capture. Probe-bound libraries were pulled down with streptavidin beads, washing occurred (removing unbound DNA), and the DNA was amplified. Then sequencing occurred (page 69, paras. 4-5). Example 2 of the reference then discusses using machine learning algorithms on sample sets, where, “Finalized models are then used to score and classify unknown samples based on the methylation of their DMRs,” (page 70, para. 3). It is noted that Examples 4 and 5 recite a similar general method in terms of sample preparation, hybrid capture, and sequencing, but do not specify particular sample types (pages 71-72).
Thus, claim 30 is directed to a judicial exception without significantly more.
Claim 44 depends on claim 30 and recites a method for providing the converted cfDNA fragments from the urine sample. This claim does not add additional judicial exceptions to the method of claim 30, and does not integrate the judicial exception of claim 30 into a practical application because there is no required active treatment step or other step that integrates the judicial exception into a practical application. See MPEP 2106.04(d). The claim does not include additional elements that are sufficient to amount to significantly more than the judicial exception because they do not amount to more than well-understood, routine, and conventional activity in view of Bierau et al. (WO 2008/155549 A2; cited in Applicant’s IDS).
Bierau teaches methods for diagnosing a urologic cancer via cell-free DNA analyses of urine samples (Abstract). Urine samples can be treated with the stabilizing agent EDTA, and this reduces DNA degradation, creates an optimal stabilizing environment during storage, and can prevent bacterial growth (page 29, para. 2). Page 9, para. 2 notes that cell-free DNA can be isolated/purified, involving separation of the cell-free DNA from cell-associated DNA (pages 7-8, joining para.). Pages 10-11, joining para., page 12, para. 2, and pages 14-15, joining para. note that isolation of the cell-free DNA can occur via a combined centrifugation and filtration process. Page 11, paras. 2-3 teach that the filter used may be a molecular weight filter that can concentrate a desired species based on molecular weight, and page 12, para. 1 notes that can have a cut of 5-10 kD. These methods, as they separate the cell-free DNA from the cellular DNA present in the sample, would naturally increase the concentration of cell-free DNA in the sample, and such a limitation is specifically stated on page 14, para. 4 of the reference. Bierau then notes that further purification of the cell-free DNA can be done so that the DNA is ready for analysis (page 15, para. 1).
Thus, claim 44 is directed to a judicial exception without significantly more for the same reasons described above for claim 30.
Claims 74 and 76-79 depend on claim 30 and provide additional details related to the trained classifier of claim 30. These claims do not add additional judicial exceptions to the method of claim 30, and also do not integrate the judicial exception of claim 30 into a practical application because there is no required active treatment step or other step that integrates the judicial exception into a practical application. See MPEP 2106.04(d). The claims do not include additional elements that are sufficient to amount to significantly more than the judicial exception because they do not amount to more than well-understood, routine, and conventional activity in view of Salhia, as described above in the rejection of claim 30.
Thus, claims 74 and 76-79 are directed to a judicial exception without significantly more for the same reasons described above for claim 30.
Claims 75 and 80-82 depend on claim 30 and narrow the structure of the bait oligonucleotides presented in claim 30. These claims do not add additional judicial exceptions to the method of claim 30, and also do not integrate the judicial exception of claim 30 into a practical application because there is no required active treatment step or other step that integrates the judicial exception into a practical application. See MPEP 2106.04(d). The claims do not include additional elements that are sufficient to amount to significantly more than the judicial exception because they do not amount to more than well-understood, routine, and conventional activity in view of Salhia, as described above in the rejection of claim 30. It is noted that claim 81 describes particular genes, and claim 82 describes particular types of cancer, and so are considered to further specify the judicial exception presented in claim 30.
Thus, claims 75 and 80-82 are directed to a judicial exception without significantly more for the same reasons described above for claim 30.
Claim 83 depends on claim 30 and requires that the cfDNA molecules be treated with bisulfite. This claim does not add additional judicial exceptions to the method of claim 30, and also does not integrate the judicial exception of claim 30 into a practical application because there is no required active treatment step or other step that integrates the judicial exception into a practical application. See MPEP 2106.04(d). The claim does not include additional elements that are sufficient to amount to significantly more than the judicial exception because they do not amount to more than well-understood, routine, and conventional activity in view of Salhia, which teaches that DNA libraries were created, bisulfite converted, and enriched with the previously created biotinylated probes used for hybrid capture in Example 1 of the reference (pages 68-69).
Thus, claim 83 is directed to a judicial exception without significantly more for the same reasons described above for claim 30.
Claims 84-86 depend on claim 30 and recite additional limitations for the bait oligonucleotides. These claims do not add additional judicial exceptions to the method of claim 30, and also do not integrate the judicial exception of claim 30 into a practical application because there is no required active treatment step or other step that integrates the judicial exception into a practical application. See MPEP 2106.04(d). The claims do not include additional elements that are sufficient to amount to significantly more than the judicial exception because they do not amount to more than well-understood, routine, and conventional activity in view of Salhia, which teaches that DNA libraries were created, bisulfite converted, and enriched with the previously created biotinylated probes used for hybrid capture in Example 1 of the reference (pages 68-69), that for CRTC1, two of the regions are hypermethylated relative to a reference genome (Table 1, page 53, note the positive DMR values and an explanation of these values on page 59, para. 5), and that that for CRTC1, three methylation sites are examined (page 53), and for KNDC1, three methylation sites are also examined (page 41), making a total of six methylation sites for these two genes.
Thus, claims 84-86 are directed to a judicial exception without significantly more for the same reasons described above for claim 30.
Claims 87-98 all depend on claim 44 and describe various limitations related to the processing of the urine sample. These claims do not add additional judicial exceptions to the method of claim 30, and also do not integrate the judicial exception of claim 30 into a practical application because there is no required active treatment step or other step that integrates the judicial exception into a practical application. See MPEP 2106.04(d). The claims do not include additional elements that are sufficient to amount to significantly more than the judicial exception because they do not amount to more than well-understood, routine, and conventional activity in view of Bierau, as described above. Additionally, the reference teaches that before methylation analyses, DNA may be amplified (page 27, para. 2, and page 37, para. 4 also notes that isolated cfDNA can be analyzed via amplification), that treated urine samples can be stored under freezing conditions (page 30, para. 3, page 32, para. 3, and page 33, para. 2), and that Amicon Ultra-15 filters can be used (e.g., page 11, para. 3, page 43, para. 3, page 44, para. 1, page 49, para. 5).
Thus, claims 87-98 are directed to a judicial exception without significantly more for the same reasons described above for claim 30.
Claim 99 depends on claim 30 and specifies the type of cancer cells that may be analyzed in claim 30. This limitation is considered to merely further specify the judicial exception presented in claim 30. This claim does not add additional judicial exceptions to the method of claim 30, and also does not integrate the judicial exception of claim 30 into a practical application because there is no required active treatment step or other step that integrates the judicial exception into a practical application. See MPEP 2106.04(d). The claim does not include additional elements that are sufficient to amount to significantly more than the judicial exception because they do not amount to more than well-understood, routine, and conventional activity.
Thus, claim 99 is directed to a judicial exception without significantly more for the same reasons described above for claim 30.
Claim 100 depends on claim 30 and describes that the subject of claim 30 is treated for their cancer. This claim does not add additional judicial exceptions to the method of claim 30, and also does not integrate the judicial exception of claim 30 into a practical application because there is no required active treatment step or other step that integrates the judicial exception into a practical application. Specifically, though a treatment step is recited, this is a general treatment step, where no specific, active, or particular treatment is recited. See MPEP 2106.04(d). The claim does not include additional elements that are sufficient to amount to significantly more than the judicial exception because they do not amount to more than well-understood, routine, and conventional activity in view of Salhia, which teaches that when examining ovarian cancer, the subject can be treated with radiation therapy and surgery to remove the cancer (page 3, para. 7).
Thus, claim 100 is directed to a judicial exception without significantly more for the same reasons described above for claim 30.
It is noted that claim 101 is not rejected under 35 USC 101, as this claim recites the use of one or more active and particular treatments.
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.
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.
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.
Claims 30, 74-80, 82-86, and 99-101 are rejected under 35 U.S.C. 103 as being unpatentable over Salhia et al. (WO 2022/178108 A1).
Salhia teaches that methods of their invention can be used to determine the methylation level of genomic regions implicated in bladder carcinomas (pages 59-60, joining para.). Page 9, para. 4 also notes that bladder cancer is included in the general term cancer. Table 1 shows target genomic regions, including regions in the genes CRTC1 (page 53) and KNDC1 (page 41), which appear in instant Tables 1-3. These genes can specifically be used to determine the presence of cancer cells present in a patient (page 21, para. 4-5). A biological sample of the invention can be cell-free genomic DNA from a urine sample (page 9, para. 2).
Salhia is primarily drawn to methods related to ovarian cancer (e.g. Abstract). Regarding ovarian cancer, on page 16, para. 4, the reference teaches that the presence of ovarian cancer can be gleaned by using hybrid capture probes, such as pull-down probes/bait sets, to selectively enrich specific DNA molecules. These probes can correlate to the genes described in Table 1, and can specifically hybridize to one or more genomic regions in a cell-free DNA sample, and specifically to differentially methylated regions, such as CpG sites, The probes can be nucleic acid molecules (page 17, para. 1).
In Example 1 of the reference, a method is described where cell-free DNA from the plasma of ovarian cancer patients and normal controls was bisulfite converted, amplified, and sequenced, and this data was then used to construct a novel classifier that used machine learning to differentiate between ovarian cancer and benign tumors (see page 68). Differentially methylated regions were identified and biotinylated RNA probes were developed, where each probe was 60-80 nucleotides in length (page 69, para. 3; instant claim 75). Then, cfDNA from plasma samples of a new cohort of patients with benign and malignant masses were taken. DNA libraries were created, bisulfite converted, and enriched with the previously created biotinylated probes used for hybrid capture (instant claims 83-84). Probe-bound libraries were pulled down with streptavidin beads, washing occurred (removing unbound DNA), and the DNA was amplified. Then sequencing occurred (page 69, paras. 4-5). Example 2 of the reference then discusses using machine learning algorithms on sample sets, where, “Finalized models are then used to score and classify unknown samples based on the methylation of their DMRs,” (page 70, para. 3). It is noted that Examples 4 and 5 recite a similar general method in terms of sample preparation, hybrid capture, and sequencing, but do not specify particular sample types (pages 71-72).
Though Salhia’s examples are focused primarily on ovarian cancer, the reference makes clear that other types of cancers, such as bladder cancers, may be examined, and that the DMRs analyzed in Table 1 of the reference can be used to evaluate said other types of cancers. Thus, prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art that the method described in the working examples (and particularly in Examples 1-2) of Salhia could be performed on samples from patients suspected of having bladder cancers, where the DMRs of Table 1 of the reference could be analyzed in the context of this cancer. Salhia shows that their sampling, training, and validation methods result in a finalized model that can score and classify unknown samples based on methylation of DMRs. This provides a non-invasive cancer diagnostic method, which would be motivating to the ordinary artisan. As the model used to determine if ovarian cancer was present in Example 1 had very high sensitivity and specificity (100% and up to 94.7%, respectively, page 69, para. 2), this method is also shown to be highly accurate, providing further motivation. As the actual sample preparation/manipulating, model development, and overall validation methods would not be changing, as the only difference in examining bladder cancer would be the starting patient population, there would be a reasonable expectation of success (instant claims 82 and 99).
Additionally, as Salhia also teaches that urine samples containing cell-free DNA may be used as biological samples in their invention, it would also be prima facie obvious to use this type of sample in the method of the working examples. Urine is even more non-invasive to obtain than plasma, which would increase patient comfort. Additionally, in either case, the same cell-free DNA is being analyzed in both sample types, and upon cfDNA isolation form the sample, the rest of the method would proceed in the same manner and produce the same results. Thus, this change in sample type would amount to simple substitution (see MPEP 2143 I (B)).
Thus, claims 30, 75, 82-84, and 99 are prima facie obvious over Salhia.
Regarding claim 74, Salhia teaches in Example 1 that in designing the trained classifier, data is based on sequence reads from the initial dataset (page 68, para. 4). Page 69, paras. 1-2 and Figure 2 show that the model then provides a probability score to determine cancerous versus non-cancerous status. Page 61, para. 4 states that utilizing the modeling methods of the invention, sequence reads can be analyzed, and methylation values for regions can be compared to threshold values. Page 65, para. 4 specifically states that probability thresholds may be used based on the methylation values obtained for the gene target regions. Page 69, para. 2 notes the use of a threshold in Example 1. Several instances in the reference also discuss normal cutoff values, where values above this cutoff are indicative of cancer (page 12, para. 4, page 57, para. 3, page 58, para. 1, and page 64, para. 2). Page 67, para. 2 states that sequence reads can be associated with methylation/computer analyses, and pages 19-20, joining para. states that sequence reads can be used to generate methylation profile information. As the methylation profile information is based on hyper- or hypomethylation (Table 1), it would be prima facie obvious that the model methods provided in Examples 1 and 2 would be based on the methylation status of the genes examined in the method (i.e. those of Table 1), and that this methylation information would be based on the sequencing data gathered during experimentation. Thus, as this modeling is used in the rejection of instant claim 30 above, it would thus be prima facie obvious that this modeling would function in the same manner when analyzing bladder cancer.
Regarding claims 76 and 78-79, Salhia teaches that their model has a defined sensitivity and specificity, as noted above and detailed on page 69, para. 2 and Figure 3. After removing false positives related to patients who did not have ovarian cancer but produced confounding results due to other related cancers/non-normal tissue, the specificity of the model was 94.7%, and the sensitivity remained 100% (page 69, para. 2 and Figure 3b). Thus, as this modeling is used in the rejection of instant claim 30 above, it would thus be prima facie obvious that this modeling would function in the same manner when analyzing bladder cancer.
Regarding claim 77, Salhia teaches in Example 2, which provides details about the model developed in Example 1, regarding their modeling methods, “Various machine learning models were tested, including random forest, C5.0 decision trees, support vector machine (SVM), generalized linear model (GLM) and gradient boosting. Models were optimized using the area under the curve (AUC) of the receiver operating characteristic (ROC) curve. More advanced models included a feature selection method prior to model construction, such as identification of differential methylation sub-regions. Finalized models are then used to score and classify unknown samples based on the methylation of their DMRs,” (page 70, para. 3). Page 61, para. 4 and page 63, para. 1 also note the various modeling methods that can be used in their instant invention. The use of the multiple modeling methods are considered to read on the mixture model described in the instant claim, as in the instant specification, paras. 185 and 244 state that a mixture model may be based on a mixture of components from underlying models. Thus, as this modeling is used in the rejection of instant claim 30 above, it would thus be prima facie obvious that this modeling would function in the same manner when analyzing bladder cancer.
Regarding claim 80, Table 1 of Salhia teaches genes taught in Table 1 of the instant specification, in addition to CRTC1 and KNDC1. These specifically include TRAPPC9, ADARB2, PON3, DENND1A, DPF3, KDM4B, NACC2, NCOR2, and SOX2-OT. As all of these genes are examined in Table 1 of Salhia, and the reference specifically notes that the genes in this table can both be examined for differentially methylated regions in cancer generally (page 59, para. 5 and page 21, paras. 3-4), and that bladder cancer can be examined (page 59, para. 6), it would be prima facie obvious that these genes could also be examined in the context of bladder cancer, though the ability of each of these genes to be useful in the trained classifier is not entirely clear – see the 35 USC 112(a) Rejection above. As claim 80 does not require that the trained classifier of claim 30 incorporate all of the genes examined with the bait oligonucleotides, these teachings are considered to read on the instant claims in accordance with the scope of enablement rejection.
Regarding claim 85, the claim notes that a plurality of the bait oligonucleotides must be related to hypermethylated regions. It is noted that this claim does not require that each bait oligonucleotide be related to hypermethylated regions due to the use of the phrasing “a plurality,” and so only requires that two or more of the bait oligonucleotides be related to hypermethylated regions. For CRTC1, Table 1 of Salhia shows that two of the regions are hypermethylated relative to a reference genome (page 53, note the positive DMR values and an explanation of these values on page 59, para. 5). Thus, in examining CRTC1 as is done in the teachings of Salhia described above in the rejection of claim 30, at least two bait oligonucleotides related to genes used to classify bladder cancer would hybridize to hypermethylated regions.
Regarding claim 86, in Table 1 of Salhia shows that for CRTC1, three methylation sites are examined (page 53), and for KNDC1, three methylation sites are also examined (page 41), making a total of six methylation sites for these two genes.
Regarding claims 100-101, Salhia teaches that when examining ovarian cancer, the subject can be treated with radiation therapy and surgery to remove the cancer (page 3, para. 7). Page 60, para. 4 also notes various cancer therapies, including surgery, radiation therapy, chemotherapy, hormone therapy, targeted therapy, and/or administration of a medication. As noted above in the rejection of instant claim 30 above, it would be prima facie obvious to use the methods of Salhia to examine bladder cancer. If a patient examined with these methods was found to have bladder cancer, it would also be prima facie obvious that said patient should be treated in order to combat the cancer. Thus, as Salhia teaches general treatments for cancer, it would also be prima facie obvious to use these methods on bladder cancer patients.
Claims 44, 87-91, and 93-98 are rejected under 35 U.S.C. 103 as being unpatentable over Salhia et al. (WO 2022/178108 A1) in view of Bierau et al. (WO 2008/155549 A2; cited in Applicant’s IDS).
Salhia teaches the methods of claims 30, 74-80, 82-86, and 99-101, as described above. However, the reference does not describe methods of treating a urine sample.
Bierau teaches methods for diagnosing a urologic cancer via cell-free DNA analyses of urine samples (Abstract). Urine samples can be treated with the stabilizing agent EDTA, and this reduces DNA degradation, creates an optimal stabilizing environment during storage, and can prevent bacterial growth (page 29, para. 2; instant claims 88-90). Page 9, para. 2 notes that cell-free DNA can be isolated/purified, involving separation of the cell-free DNA from cell-associated DNA (pages 7-8, joining para.). Pages 10-11, joining para., page 12, para. 2, and pages 14-15, joining para. note that isolation of the cell-free DNA can occur via a combined centrifugation and filtration process (instant claim 91). Page 11, paras. 2-3 teach that the filter used may be a molecular weight filter that can concentrate a desired species based on molecular weight, and page 12, para. 1 notes that the molecular weight can have a cut of 5-10 kD (instant claim 93). These methods, as they separate the cell-free DNA from the cellular DNA present in the sample, would naturally increase the concentration of cell-free DNA in the sample, and such a limitation is specifically stated on page 14, para. 4 of the reference. Bierau then notes that further purification of the cell-free DNA can be done so that the DNA is ready for analysis (page 15, para. 1; instant claim 44).
Bierau generally teaches that, “the sensitivity obtained with the cell-free DNA fraction from urine is higher than the sensitivity obtained with the traditionally used sediment fraction. The use of the cell-free DNA fraction thus leads to an improvement in the sensitivity and specificity of tumor marker detection in urine for cancers, in particular those cancers that release their cells and cellular components directly in the urethra,” (page 4, para. 3).
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use the teachings of Bierau to manipulate the urine sample of Salhia before performing the cfDNA analysis. Bierau highlights the usefulness of detecting cfDNA in urine, particularly in relation to cancers, which would make the reference generally of use to the ordinary artisan performing the method of Salhia described above. Then, the ordinary artisan would specifically be motivated to use the filtration methods described by the reference in order to concentrate the cfDNA and eliminate unwanted products – this would increase the overall yield of the method of Salhia, thereby potentially improving the accuracy of the method in terms of correctly diagnosing patients, which would improve patient outcomes. There would be a reasonable expectation of success as the methods of Bierau result in cfDNA that is ready for further analysis, and particularly for analysis related to cancer, and so once the extracted cfDNA is obtained, the method of Salhia can proceed as already described in this latter reference.
Thus, claims 44, 88-91, and 93 are prima facie obvious over Salhia in view of Bierau.
Regarding claim 87, Bierau teaches that before methylation analyses, DNA may be amplified (page 27, para. 2), and page 37, para. 4 also notes that isolated cfDNA can be analyzed via amplification. It would be prima facie obvious to amplify the isolated cfDNA before performing the analysis methods of Salhia, as this would mean the sample would have a greater number of copies of the target genes to detect. As the methods of Salhia are drawn to using methylation levels of gene targets to detect cancer, having additional target sequence copies present would increase the accuracy of the results determined by Salhia, which in turn would provide more accurate diagnostic information. As Salhia is drawn to cancer-related methods, this increased accuracy would be of interest to both clinicians and patients.
Thus, claim 87 is prima facie obvious over Salhia in view of Bierau.
Regarding claim 94, in the working examples on pages 51-54, Bierau generally describes taking a urine sample and using a portion of it for analysis, and analyzes the amount of DNA present in portions of the sample available for said analysis. Figure 4A shows the relevant results, where compared to whole urine, many more copies of DNA were obtained in the supernatant and pellet portions of the urine sample. In these examples, 45 mL of urine was then spun down into a pellet and 300 µL of supernatant. This naturally significantly lowers the volume in which the DNA is existing by more than 2-fold, and so naturally would also increase the concentration of the DNA present in the pellet/supernatant by more than 2-fold, as shown in Figure 4A. Thus, this example shows that the methods of Bierau are capable of meeting the concentration limitations presented in instant claim 94. It would also be prima facie obvious to the ordinary artisan to follow the general guidelines for sample and final volume used by Bierau, as these are shown to produce successful results in terms of capability of recovering and analyzing DNA.
Thus, claim 94 is prima facie obvious over Salhia in view of Bierau.
Regarding claims 95-96, similar to in instant claim 94, in the working examples of Bierau described on pages 51-54, 45 mL samples were mixed with 5.8 mL of stabilizing buffer. This results in 50.8 mL of treated urine sample (instant claim 96). This treated sample was then spun down to a pellet and 300 µL, and thus the final volumes are an incredibly small fraction of the initial treated urine sample (instant claim 95). As noted above in the rejection of instant claim 94, it would be prima facie obvious to the ordinary artisan to follow the general guidelines for sample and final volume used by Bierau, as these are shown to produce successful results in terms of capability of recovering and analyzing DNA.
Thus, claims 95-96 are prima facie obvious over Salhia in view of Bierau.
Regarding claim 97, Bierau teaches that treated urine samples can be stored under freezing conditions (page 30, para. 3, page 32, para. 3, and page 33, para. 2). Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to perform this freezing in the event that a sample needed to be stored for a long period of time – for example, if samples collected from different time points were going to be mass analyzed, it would be advantageous to freeze each sample, transport them to a single facility, and manipulate each sample at a single location, as this would be a resource-efficient manner in which to analyze samples. As freezing can easily be accomplished (e.g. with the use of a freezer), and Bierau teaches that frozen samples can still later be analyzed, there would be a reasonable expectation of success.
Thus, claim 97 is prima facie obvious over Salhira in view of Bierau.
Regarding claim 98, on page 29, para. 2 of Bierau the reference states that the use of the stabilizing buffer allows for the maintaining of the sample at room temperature for 2-3 days and removes the need for centrifugation equipment at the collection site. Thus, it would be prima facie obvious to add the stabilizing buffer containing EDTA described by Bierau above as soon as possible after collecting a sample to prevent the sample from degrading at room temperature and to take advantage of the lack of need for immediate centrifugation. As the reference teaches that centrifugation normally occurs within 4 hours of obtaining a urine sample, the ordinary artisan would recognize that the buffer would need to be added before this point. This would overlap with the time range described for the treating in the instant claim.
Thus, claim 98 is prima facie obvious over Salhira in view of Bierau.
Claim 81 is rejected under 35 U.S.C. 103 as being unpatentable over Salhia et al. (WO 2022/178108 A1) in view of Larsen et al. (Int. J. Mol. Sci., 2019).
Salhia teaches the methods of claims 30, 74-80, 82-86, and 99-101, as described above. However, the reference does not teach any of the genes described in instant claim 81.
Larsen teaches methylation-based biomarkers related to bladder cancers (Abstract). Figure 1 shows that TWIST1 is DNA methylation biomarker used in bladder cancer. Table 1 and Table 2 also notes DNA-methylation biomarkers in association with bladder cancer as are used in the art, and also recites TWIST1.
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use the teachings provided by Larsen to add at least TWIST1 as an additional biomarker to the teachings of Salhia that are described above in the rejection of instant claim 30. Particularly, Larsen provides biomarkers already successfully used in the prior art in relation to bladder cancer, and for TWIST1, the sensitivity associated with the use of this biomarker is very high (Figure 1). Thus, the ordinary artisan would be motivated to add this DNA-methylation biomarker to the methods of Salhia to improve the sensitivity, and therefore accuracy, of the trainer and the overall diagnostic method. As this would not be altering the actual method steps of Salhia, and would simply be adding an additional biomarker, where the biomarker has already been successfully used in similar contexts, there would be a reasonable expectation of success.
Therefore, claim 81 is prima facie obvious over Salhia in view of Larsen.
Claim 92 is rejected under 35 U.S.C. 103 as being unpatentable over Salhia et al. (WO 2022/178108 A1), in view of Bierau et al. (WO 2008/155549 A2; cited in Applicant’s IDS), and further in view of Biocompare (“Amicon Ultra-15 Centrifugal Filter Units from Millipore,” 2007).
Salhia in view of Bierau teaches the method of claims 44, 87-91, and 93-98, as described above. Bierau teaches that in pages 14-15, joining para. that the filter is capable of capturing cfDNA. Throughout the reference and in the working examples of Bierau, it is taught that Amicon Ultra-15 filters can be used, and specifically notes the 5K version (e.g., page 11, para. 3, page 43, para. 3, page 44, para. 1, page 49, para. 5).
Biocompare discusses the specifics of these filters. After choosing a molecular weight cut off based on the desired target, the reference states, “To use the filter units, you first place your sample in the filter unit sample reservoir. The tube is then centrifuged and the liquid remaining (retentate) in the reservoir is composed primarily of molecules larger than the cut-off while molecules below the molecular cut-off pass through the filter,” (para. 1). Thus, Biocompare teaches that with these filters, the liquid filtrate contains unwanted molecules that pass through the filter, while the filter captures the target molecules.
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious to one of ordinary skill in the art using the teachings of Biocompare and Bierau that the Amicon filters of Bierau can capture cfDNA in the filter, based on molecular weight, and that this size exclusion can aid in purifying the cfDNA from the unwanted components of the urine sample. As the purpose of the urine manipulations of Bierau is to concentrate the cfDNA for later analysis, it would thus be prima facie obvious to choose the filter size in such a way that the cfDNA is captured but most of the other portions of the urine sample, including salts, pass through the filter. This would increase the purity of the resulting cfDNA sample, which would increase the overall yield of the method of Salhia, thereby potentially improving the accuracy of the method in terms of correctly diagnosing patients, which would improve patient outcomes. As Bierau teaches a variety of molecular weights that may be used in their methods (see page 12, para. 1, page 32, para. 1), and the Amicon filters are well known in the art and come in a variety of sizes, as shown in Biocompare, the ordinary artisan would be capable of choosing the size that would result in the greatest concentration of cfDNA and the most passage of unwanted other urine elements.
Thus, claim 92 is prima facie obvious over Salhia, in view of Bierau, and further in view of Biocompare.
Conclusion
No claims are currently allowable.
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/F.F.G./Examiner, Art Unit 1681
/SAMUEL C WOOLWINE/Primary Examiner, Art Unit 1681
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