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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 3/18/2026 has been entered.
Applicant’s arguments and amendments have been thoroughly reviewed and considered. Claim 25 has been canceled. Claims 27-34 have been added. Claims 1-16, 18-24, and 26-34 are pending and are examined on the merits herein.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 3/18/2026 is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Response to Applicant’s Amendments
Claim Objections
Claim 1 was objected to due to minor informalities. In light of Applicant’s amendments to the claims submitted 3/18/2026, this objection has been withdrawn, but see new grounds of objection below.
35 USC 112(b) Rejections
Claims 1-16 and 18-26 were rejected for various indefiniteness issues. In light of Applicant’s amendments to the claims submitted 3/18/2026, these rejections have been withdrawn for all currently pending claims. Claim 25 has been canceled, so this rejection has been rendered moot. See also new grounds of rejection below.
35 USC 112(d) Rejections
Claim 25 was rejected for failing to further limit the claim upon which it depended. As this claim has been canceled, this rejection has been rendered moot.
35 USC 103 Rejections
Claims 1-16 and 18-26 were rejected under 35 U.S.C. 103 as being unpatentable over Saffroy et al. (US 2016/0319365 A1), in view of Carson et al. (US 2010/0086918 A1), and in view of Bramlett et al. (US 2016/0194694 A1) and various combinations of references. In light of Applicant’s amendments to the claims submitted 3/18/2026, these rejections have been withdrawn for all currently pending claims. However, see new grounds of rejection and “Response to Applicant’s Arguments” below. Claim 25 has been canceled, and so this rejection has been rendered moot.
Response to Applicant’s Arguments
Regarding the 35 USC 103 Rejections previously presented in the Final Rejection, Applicant argues that the cited references do not teach the limitation of newly amended step (f) in claim 1, and states that the instant invention provides technical advantages and unexpected results over the cited references (Remarks, pages 11-12).
The combination of Saffroy, in view of Carson, and in view of Bramlett does not teach the use of replicate samples, and so the Examiner agrees that the references do not fully teach step (f) of claim 1, where each of three or more replicates is used to quantify relative abundance of DNA. This is the basis for the new grounds of rejection presented below.
Regarding Applicant’s alleged technical advantages and unexpected results, Applicant appears to merely point out embodiments of their invention where multiple replicates were used. Applicant does not point out in these uses what would constitute a technical advantage or unexpected result over the prior art, other than stating that their methods are “improved methods for quantifying DNA gene fusion molecules using three or more replicate samples comprising allelic fraction replicates,” (Remarks, pages 12-13). It appears that Applicant is arguing that the act of using replicate samples alone provides “improved” advantages/results, but the specificity of said improvements is not stated. Applicant is directed to MPEP 716.02 for the requirements needed to show that their claimed invention produces unexpected results, and particularly is directed to MPEP 716.02(d), which states that an unexpected results must be commensurate in scope with the claimed invention. The citations provided by Applicant in their Remarks that concern working examples of their invention focus on specific gene fusions and/or allele fractions that are not specifically claimed in instant claim 1. Additionally, the use of replicate samples and their associated benefits is well-known in the prior art – see the new grounds of rejection in the 35 USC 103 Rejections section below.
Thus, while Applicant’s amendments to the claims require new grounds of rejection, Applicant’s arguments are not persuasive to obviate the use of the references previously provided in the Final Rejection. The relevant portions of these references and their combinations are reiterated below.
Claim Objections
Claim 26 is objected to because of the following informality: as each allelic fraction is simply a number, the phrase “three or more replicate samples comprises” should read “three or more replicate samples is one of” or similar wording. Appropriate correction is required.
Claim 29 is objected to because of the following informality: the claim should read “The method of claim 1, wherein step (a) comprises dividing the test sample….,” as this claim simply further narrows the number of replicate samples used, and does not describe a new method step. Appropriate correction is required.
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 33 and 34 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.
Claim 33 requires the production of three or more ratios for each replicate of the three or more replicates of claim 1. In claim 1, step (f) requires that each replicate be used with the reference sample to produce a single ratio. It is unclear what data a second and third ratio for each replicate would be derived from. Claim 33 states these ratios are in part created based on step (e) of claim 1, but this step does not recite any quantification of ratios. Therefore, the scope of the claim is indefinite. The claim will be interpreted as though each replicate must produce its own ratio in step (f) of claim 1.
Claim 34 is rejected based on its dependence on rejected claim 33.
Claim Interpretation
In claim 1, there is no requirement that different allelic fractions be used with each replicate, nor is the actual allelic fraction information for a given replicate used in the method. Thus, a single allelic fraction may be used for all replicates (though see dependent claims 27-28, which do specify that allelic fractions are different or the same from one another for the replicates). This allele fraction limitation will be considered met if the prior art teaches, alone or in combination, a sample involving any amount of a gene fusion, as this sample will inherently have an allelic fraction.
In claim 26, which specifies particular amounts of allelic fraction that must be used. This claim also does not state if the allelic fractions must be the same or different for each sample, and so either scenario is considered to be encompassed by the claim.
It is also generally noted in the claims that the allelic fraction in the replicate samples is not explicitly tied to the gene fusion actually examined in the method of claim 1.
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 1-10, 13-16, 20-24, 29-30, and 32-33 are rejected under 35 U.S.C. 103 as being unpatentable over Saffroy et al. (US 2016/0319365 A1), in view of Carson et al. (US 2010/0086918 A1), in view of Bramlett et al. (US 2016/0194694 A1), and in view of Salzman et al. (PLoS Biology, 2011; cited in Applicant’s IDS).
Regarding claim 1, Saffroy teaches a method for detecting chromosomal rearrangements between different chromosomal regions in a biological sample of a human subject (Abstract). Said sample can be cell-free tumor DNA in the blood (paras. 12, 62, and 70). The method involves using multiplex PCR (specifically noted to include a polymerase in para. 113) to amplify the target molecules with sets of primers, including forward and reverse primers (paras. 25 and 34-36, and Figure 1). Figure 1 shows at least six forward and six reverse primers, where the primers tile across the target regions, and paragraph 36 specifically notes these primers are specific to a reference chromosomal region. These reference regions are those present in the typical human genome (para. 37). Saffroy specifically teaches that more than one set of primers can be used, and that each set contains a plurality of primers, comprising at least two primers (paras. 34-36 and 90). Therefore, at least 20 forward and reverse primers could be used. The chromosomal rearrangement examined can be translocations, meaning the different chromosomal regions can be on different chromosomes (paras. 37 and 42). Figure 1 shows that after amplification, fusion amplicons are produced. Saffroy also teaches that using cell-free DNA from blood can be used for detecting mutations or gene rearrangements, diagnosis, and/or prognosis (paras. 12 and 18). This reference notes that that liquid blood samples can contain circulating tumor DNA (paras. 62 and 70). Saffroy then teaches that chromosomal rearrangements are associated with cancers, particularly non-small cell lung cancer (e.g. paras. 2-4). It would therefore be prima facie obvious that if chromosomal rearrangements associated with cancer were found in the cell-free DNA blood samples (which contain circulating tumor DNA), that the ordinary artisan could conclude that the subject contained tumor cells that also contained the chromosomal rearrangement. Saffroy also teaches that the method can further comprise sequencing the amplification products (para. 137). Lastly, Saffroy teaches performing the method on negative controls that do not contain chromosomal rearrangements along with the target samples (paras 218).
However, Saffroy does not specifically teach that these controls are a different chromosomal region than the target DNA, and also does not teach methods of quantifying the relative abundance of the fusion molecules in the test sample or the use of multiple replicate samples.
Carson teaches multiplex PCR methods for detecting variant polynucleotides that differ from wild-type polynucleotides (Abstract), where said variation can be a fusion of nucleotides (para. 6). This reference also teaches that variant polynucleotides can be determined and relatively quantified via plotting signal intensity ratios of variant:wild-type PCR products (para. 75 and Figures 5A-5B). Carson also teaches these ratios in competitive PCR – where the amount of the target is not known. Here, known amounts of “competitor DNA” are mixed with the target sample, PCR is performed, and then the ratios of target:competitor DNA are analyzed (para. 89). This can then be used to estimate the approximate concentration of target DNA.
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 relative quantification methods of Carson in the method of Saffroy. Carson specifically teaches that these methods can be used when the target amount of DNA is not known, and can lead to determinations of both relative and approximate absolute abundance of target DNA. This would be particularly useful in the cancer applications of Saffroy, as particular thresholds of concentrations for circulating tumor DNA may aid in diagnosis and prognosis determinations. Additionally, as Saffroy does not teach quantification methods, adding those of Carson would only add steps to the method of Saffroy without changing any other existing steps. The results would also be predictable, as the method of Saffroy ends with sequence reads produced, and so the amount of these reads could be determined and then used in the manner described by Carson.
However, Carson specifically teaches finding ratios between target sequences and wild-type sequences, and thus does not teach a reference primer pair that amplifies a different region than those amplified by the forward and reverse fusion primers. Regarding replicate samples, Figure 2 of Carson notes an array with “triplicate spots,” but the reference provides no additional details regarding what constitutes said triplicates.
Bramlett teaches multiplex amplification occurring in a single reaction mixture, where the target polynucleotide can be a fusion sequence (Abstract and para. 234). This reference teaches a specific method in which reference samples are used, where reference primers amplify a reference sequence while gene fusion primers amplify a target sequence in the same sample (para. 300). Bramlett also teaches that more than one reference sequence can exist (e.g. those sequences in the “reference list”; para. 263), and in similar and more general teachings of this method, the reference and test sample can be from different transcripts (paras. 286, 289, and 301). This method can be used to measure the abundance of target gene fusion transcripts.
Prior to the effective filing date of the claimed invention, it also would have been prima facie obvious for one of ordinary skill in the art to use the teachings of Bramlett to inform the controls of the method of Saffroy in view of Carson. Specifically, this would result in the use of a reference sample in the reaction mixture that differs from the sequence of the target sample, along with reference primer pairs similar in structure to the forward and reverse primers described by Saffroy to amplify the reference sample. Bramlett teaches that these methods can aid in finding the abundance of gene fusions in a sample, and so aligns with the context of the method of Saffroy in view of Carson. Since the reference sequence of Bramlett is already known, as evidenced by the taught reference list, the ordinary artisan would have a reasonable expectation of success in being able to design appropriate reference primers and detecting this reference sequence. The results would also be predictable, as this combination would end with quantification of both target and reference reads.
However, Bramlett does not teach the use of replicate samples.
Salzman teaches an analysis of gene fusion events related to ovarian carcinoma (Abstract). The reference analyzed tumor mRNA to detect diagnostic gene fusion events utilizing RT-PCR and sequencing methods (pages 6-7, “RNA-SEQ Library Preparation” and “RT-PCR Validation and Screening”). With regard to RT-PCR methods, Salman states, “It should be noted that, in order for a patient sample to be called fusion-positive, we required that the majority of technical PCR replicates be positive…,” (page 2, column 2, para. 5). Page 7, column 1, para. 3 notes that for each sample that underwent RT-PCR, up to six technical replicates were used, where the amount of replicates that were shown to be fusion-positive was used to determine if a sample itself was considered fusion-position.
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 guidance provided by Salzman to incorporate replicate samples into the method of Saffroy, in view of Carson, and in view of Bramlett to arrive at the method of claim 1. Specifically, Salzman teaches that replicate samples are useful when analyzing gene fusions, as they allow for the determination of whether a sample is truly positive for a fusion, which can have diagnostic implications. As the methods of Saffroy are also focused on gene fusions and cancer, this would be motivation to combine the teachings of Salzman with that of Saffroy, in view of Carson, and in view of Bramlett. There would be a reasonable expectation of success as Salzman specifically teaches the use of replicates with a single patient sample, and is able to perform PCR amplification on said replicates. Additionally, as Salzman teaches the use of up to 6 replicate samples, where these replicates were specifically used to determine the fusion-positive status of a patient sample, the ordinary artisan would recognize that this number would be sufficient to utilize in their own analyses. Less replicates could impact data accuracy, while more replicates could be taxing in terms of time, efficiency, and use of resources. Thus, the ordinary artisan would be motivated and capable of performing the method of Saffroy, in view of Carson, and in view of Bramlett on up to 6 replicate samples, as is taught in Salzman. This would involve performing the amplification, sequencing, and analysis methods (including comparing sequencing data to that of the reference sample) for each replicate.
As noted above in the “Claim Interpretation” section, only a single allelic fraction need be used in the claimed method, where the allelic fraction need not be specified. The method of Saffroy, in view of Carson, in view of Bramlett, and in view of Salzman utilizes multiple replicate samples and identifies genomic fusions in said samples – where each sample will naturally have an allelic fraction for said fusion. These references in combination thus meet every limitation of instant claim 1
Therefore, claim 1 is prima facie obvious over Saffroy, in view of Carson, in view of Bramlett, and in view of Salzman.
Regarding claims 2-5, Carson teaches that by tracking and comparing the occurrence of gene fusions over time in a population of cells in a subject, the susceptibility of said subject to disease, including diagnosis and progression monitoring, can be determined. This is specifically taught for cancer (para. 9). Carson also teaches monitoring the progression of cancer by explicitly measuring multiple types of sequence variations, such as translocations and deletions, over time by comparing results of an original assay to a subsequent assay (paras. 53 and 54). This can also be used to monitor treatment efficacy if affected chromosomal regions appear to have stabilized between assays (para. 53). Though Carson does not provide a specific number of time points that can be analyzed, it is clear that two or more may be used, due to the example provided at the end of paragraph 54 and the use of the phrase “quantitating the number of cells in successive cell samples which bear and acquire the deletion or other polymorphism at separate locations in the body and/or over time,” (emphasis added).
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 time point measurements of Carson in the method of Saffroy, in view of Carson, in view of Bramlett, and in view of Salzman. Specifically, these measurements can be used to compare ratios over two time points (instant claim 2), compare ratios between time points to determine the efficacy of a cancer treatment (instant claim 3), compare ratios over successive time points totaling three or more during a subject’s cancer progression (claim 4), and compare ratios of multiple types of sequence variations (instant claim 5). The ordinary artisan would be motivated to do this because it could aid in diagnosis and outcomes for cancer patients (which are already examined in the method described above in the rejection of claim 1). There would be a reasonable expectation of success in employing these methods because they would simply involving performing the method of Saffroy, in view of Carson, in view of Bramlett, and in view of Salzman described for claim 1 on multiple samples at multiple time points. The actual steps of this method would remain unchanged.
Thus, claims 2-5 are prima facie obvious over Saffroy, in view of Carson, in view of Bramlett, and in view of Salzman.
Regarding claim 6, Saffroy teaches chromosomal rearrangements involving ALK and ROS1 (paras. 46-47). Specifically, primers for ALF/ELM4 (para. 92) and ROS1/CD74 or EZR or SLC34A2 chromosomal rearrangements (para. 98) are recited.
Regarding claims 7-10 and 24, as noted above, the method of Saffroy alone teaches multiple sets of primers that each contain a plurality of primers (paras. 34-36 and 90). It would therefore be prima facie obvious to include multiple sets of reference primers, and therefore multiple reference primer pairs, in the method of Saffroy, in view of Carson, in view of Bramlett, and in view of Salzman, where each primer pair can amplify a different reference sequence. This would then lead to a plurality of reference amplicons corresponding to the different reference sequences, as well as sequence reads corresponding to the different reference regions, which could then be used when quantifying the relative abundance of the fusion molecules. This also aligns with the teachings of Bramlett that recite multiple reference sequences (para. 263). Additionally, Saffroy teaches DNA molecules with lengths less than 200 base pairs, which overlaps with the amplified region length of 40-160 base pairs described in claims 7 and 24 (paras. 83-84). Saffroy also teaches that cell-free DNA fragments are typically 70-200 base pairs long, which also overlaps with the 40-160 base pair length described in claim 24 (para. 12). MPEP 2144.05 I states, “In the case where the claimed ranges "overlap or lie inside ranges disclosed by the prior art" a prima facie case of obviousness exists. In re Wertheim, 541 F.2d 257, 191 USPQ 90 (CCPA 1976).” As this 40-160 base pair length range is not described as critical or producing unexpected results in the instant specification, a prima facie case of obviousness exists to amplify DNA regions of 200 or less with all primer pairs (i.e. fusion forward, fusion reverse, and reference) in the method of Saffroy, in view of Carson, in view of Bramlett, and in view of Salzman. This would thus arrive at the methods of instant claims 7 and 24. Regarding instant claim 8 specifically, given that the range for cell-free DNA fragments taught by Saffroy is 70-200 base pairs, it would be prima facie obvious that the regions amplified by reference primer pairs could be different lengths within that range, and so could differ in length from one another by at least five nucleotides. Regarding instant claims 9 and 10 specifically, as Saffroy teaches that sets of primers can contain at least two primers, it would be prima facie obvious that the sets of reference primers could contain at least 10 primer pairs (or 10-30 primer pairs).
Regarding claims 13-14, Bramlett teaches that sequence reads from targets of interest can be compared to reference sequences and reference lists (para. 263). Additionally, Bramlett teaches that “reads that align to the reference sequences that do not correspond to the one or more sequences of interest can be retained or discarded,” (para. 263). This reference also teaches that primers can be selected to reduce GC bias and low melting temperatures (para. 448). It would thus be prima facie obvious for one of ordinary skill in the art to eliminate outlier reference sequence reads that may align with reference regions but are not of interest in the method of Saffroy, in view of Carson, in view of Bramlett, and in view of Salzman (instant claim 13). By eliminating these unwanted reads, it would provide cleaner data with which to make ratios and comparisons with gene fusion target data. The ordinary artisan would also be motivated to design primers based on the factors described by Bramlett, such as GC content. This would include the design of the reference primers. Primer design would then affect the reference regions that are amplified. If the GC content of reference sequence reads did not then correspond to expected values, these reference sequence reads could be considered outliers, and could then be discarded, as taught by Bramlett (instant claim 14).
Thus, claims 13-14 are prima facie obvious over Saffroy, in view of Carson, in view of Bramlett, and in view of Salzman.
Regarding claims 15-16, in the specific method taught by Saffroy with regard to the rejection of claim 1 above, the target DNA molecule is X base pairs long, and the primers are spaced apart at a distance of less than X/2 (paras. 34-36 and 80). X is preferably less than or equal to 600, and can be 200 base pairs or less (paras. 83-84). If X is 200 base pairs, then the primers would be spaced at intervals of less than 100 base pairs, given the teachings of paragraphs 34-36.
Regarding claims 20 and 21, Saffroy teaches Table 1, in which signal intensities for samples and controls are reported. Carson also teaches several graphical representations of their methods, including Figure 2 which plots tumor:wild-type intensity along the length of the genome, as well as Figure 5A, which plots mutation:wild-type ratios at different locations. Salzman shows in Figure 2 graphical representations of copy number variations in a ratio of tumor:normal tissue. The combinations of these teachings would make it prima facie obvious for the ordinary artisan to use similar illustrative methods for the method of Saffroy, in view of Carson, in view of Bramlett, and in view of Salzman. Specifically, the ordinary artisan could use the teachings of Saffroy, Carson, and Salzman to make a table of results containing the ratios of fusion molecules to reference molecules at different time points, and then graph this information. The information for the table and graph would already be available to the ordinary artisan after performing the method of Saffroy, in view of Carson, in view of Bramlett, and in view of Salzman, so it would be logical to present this data in a way that is accessible to other researchers, medical professionals, and even patients when in a cancer setting. Creating reports and graphs is well-known in the art, as evidenced by Saffroy, Carson, and Salzman.
Thus, claims 20-21 are prima facie obvious over Saffroy, in view of Carson, in view of Bramlett, and in view of Salzman.
Regarding claim 22, the method of Saffroy of paragraphs 34-36 specifically notes that the DNA molecules of the biological sample are amplified via multiplex PCR. This multiplex PCR specifically means that more than two molecules are amplified and more than two primer sets are used (para. 112). The primers used for the multiplex PCR are designed to amplify the genomic rearrangement (i.e. the gene fusion) of interest (para. 104). Saffroy also teaches that using cell-free DNA from blood can be used for detecting mutations or gene rearrangements, diagnosis, and/or prognosis (paras. 12 and 18). This reference notes that that liquid blood samples can contain circulating tumor DNA (paras. 62 and 70). Finally, Saffroy teaches that chromosomal rearrangements are associated with cancers, particularly non-small cell lung cancer (e.g. paras. 2-4). It would therefore be prima facie obvious that if chromosomal rearrangements associated with cancer were found in the cell-free DNA blood samples (which contain circulating tumor DNA), that the ordinary artisan could conclude that the subject contained tumor cells that also contained the chromosomal rearrangement.
Regarding claim 23, as taught in the rejection of claim 22, multiplex PCR is used in the method of Saffroy, in view of Carson, in view of Bramlett, and in view of Salzman. By using multiple primer sets and creating multiple amplicons, multiple sequence reads corresponding to said different amplicons would be created. As the method of Saffroy, in view of Carson, in view of Bramlett, and in view of Salzman teaches comparing the reads of a reference sequence to that of an individual target sequence, it would be prima facie obvious for the ordinary artisan to analyze this multiplex data by noting the number of sequence reads for each different amplicon and comparing it to that of the reference sequence, creating multiple ratios. The sequence data would be readily available, and this would simply involve performing the ratio analytical method described by Saffroy, in view of Carson, in view of Bramlett, and in view of Salzman multiple times. This would not change any core aspect of the gene fusion amplification method.
Regarding claims 29-30, as noted above in the rejection of claim 1, Salzman teaches the use of up to 6 replicate samples per test sample, and the use of said replicates is prima facie obvious in the method of Saffroy, in view of Carson, in view of Bramlett, and in view of Salzman, where each sample is separately analyzed via amplification and sequencing methods. This would naturally produce up to six distinct reaction mixes, with one reaction mix for each replicate.
Regarding claim 32, as noted above in the rejection of claim 1, Saffroy teaches the use of sequencing methods (para. 137), but the specific type of sequencing to be used is not limited. Bramlett teaches the use of next-generation sequencing methods after amplification, and provides examples of commercially available platforms for doing so (para. 309). Salzman also teaches the use of Illumina sequencing via a paired-end protocol and flow cells methods (page 6, column 2, para. 4 and page 7, column 1, para. 7), where the latter method is specifically taught as being high-throughput.
As Saffroy does not require any particular type of sequencing in their methods, it would be prima facie obvious to use next-generation sequencing methods, as exemplified by Bramlett and Salzman, in the method of Saffroy, in view of Carson, in view of Bramlett, and in view of Salzman. These methods are well-known, as evidenced by Bramlett and Salzman, and are compatible with amplification methods. These methods are also high-throughput, which allows more nucleic acid molecules to be sequenced more quickly compared to other sequencing methods, saving time during analysis.
Thus, claim 32 is prima facie obvious over Saffroy, in view of Carson, in view of Bramlett, and in view of Salzman.
Regarding claim 33, as noted above in the rejection of claim 1, in Saffroy, in view of Carson, in view of Bramlett, and in view of Salzman, each replicate sample goes through the entirety of the method of Saffroy, in view of Carson, and in view of Bramlett. This would include obtaining a ratio of the reads of the gene fusion data in the replicate sample as compared to that of the reference sample for each replicate. Thus, Saffroy, in view of Carson, in view of Bramlett, and in view of Salzman meets the method of claim 33.
Claims 11-12 and 34 are rejected under 35 U.S.C. 103 as being unpatentable over Saffroy et al. (US 2016/0319365 A1), in view of Carson et al. (US 2010/0086918 A1), in view of Bramlett et al. (US 2016/0194694 A1), in view of Salzman et al. (PLoS Biology, 2011; cited in Applicant’s IDS), and further in view of Cantor et al. (US 9,605,313 B2).
Regarding claims 11-12, Saffroy, in view of Carson, in view of Bramlett, and in view of Salzman teach the methods of claim 7, as described above. Additionally, as taught above in the rejection of claims 13-14, Bramlett teaches methods for eliminating particular reference sequence reads.
However, none of these references teach taking a median number of sequence reads.
Cantor teaches methods for assessing genetic variations (Abstract). This includes gene fusions (column 60, para. 4). When processing data, Cantor teaches that reference median counts from a known set of references can be calculated and uninformative reference sample sections can be removed (column 50, 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 incorporate the teachings of Cantor into the method of Saffroy, in view of Carson, in view of Bramlett, and in view of Salzman. This would lead to calculating the median number of reference sequence reads from the multiple reference amplicons, and then using this value in the ratio with the number of fusion molecule sequence reads. This would be a way to incorporate the count of each reference amplicon in a way that would not be skewed if unfiltered outliers were present, such as would be the case if the mean was taken. By incorporating values from multiple reference regions, it can also further highlight any abnormalities in the target sample, which may aid in diagnosis of a subject. Additionally, the ordinary artisan would be motivated to eliminate unwanted reads when performing this analysis, as taught in Bramlett and Cantor. By eliminating said unwanted reads, it would provide cleaner data with which to make ratios and comparisons with gene fusion target data. There would be a reasonable expectation of success incorporating these teachings into the method of Saffroy, in view of Carson, in view of Bramlett, and in view of Salzman because the primer and amplification methods would be unchanged – this would simply add data processing step(s) to the method. Additionally, calculating medians and removing unwanted data is well-known in the art, as evidenced by Cantor and Bramlett.
Therefore, claims 11-12 are prima facie obvious over Saffroy, in view of Carson, in view of Bramlett, in view of Salzman, and further in view of Cantor.
Regarding claim 34, Saffroy, in view of Carson, in view of Bramlett, and in view of Salzman teach the method of claim 33, as described above. Cantor additionally teaches that data processing methods can involve mean calculations, particularly for normalized counts of sequence read data or processed products thereof (columns 47-48 joining para.). In Example 2, the reference teaches calculating the mean number of copies of DNA under different conditions (column 83, column 1, para. 4).
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 guidance provided by Cantor in the method of Saffroy, in view of Carson, in view of Bramlett, and in view of Salzman to average the ratios for each replicate sample to produce a single ratio. Cantor provides a context similar to that of the claimed invention (examining sequence reads associated with genetic variations, including gene fusions), and notes that averaging sequencing read data products is possible. Provided that each replicate sample was taken and processed in a similar manner Saffroy, in view of Carson, in view of Bramlett, and in view of Salzman, the results produced by each replicate should be similar to one another, with slight variation accounting for chance, error, noise, etc. Providing an average ratio value accounting for all the various replicate:reference ratios would allow for the evaluation of the variation within the replicates to see if said variation is as predicted. Particularly large variation could indicate peculiarities associated with a particular replicate that may need to be further examined. This would increase accuracy and confidence in results, which would be motivating to the ordinary artisan. There would be a reasonable expectation of success as calculating averages is a simple mathematical process that would be possible for the ordinary artisan.
Thus, claim 34 is prima facie obvious over Saffroy, in view of Carson, in view of Bramlett, in view of Salzman, and further in view of Cantor.
Claims 18-19 are rejected under 35 U.S.C. 103 as being unpatentable over Saffroy et al. (US 2016/0319365 A1), in view of Carson et al. (US 2010/0086918 A1), in view of Bramlett et al. (US 2016/0194694 A1), in view of Salzman et al. (PLoS Biology, 2011; cited in Applicant’s IDS), and further in view of Nacu et al. (BMC Medical Genomics, 2011).
Regarding claims 18-19, Saffroy, in view of Carson, in view of Bramlett, and in view of Salzman teach the method of claim 1, as described above. Additionally, this combination of references teaches taking measurements at different time points, as described above in the rejection of claims 2-5.
Nacu teaches measuring expression levels of gene fusion events associated with human prostate adenocarcinoma (Abstract). These gene fusion expression events were measured with replicate samples and through the calculation of standard errors (Figure 3C caption). Nacu then put this information into a graph to compare the different expression levels and draw conclusions (pages 8-9 through to “Tissue Specificity of TICs”).
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 Nacu to inform the methods of Saffroy, in view of Carson, in view of Bramlett, and in view of Salzman to arrive at the invention of claims 18-19. Specifically, this would involve using the replicate samples and replicate ratio values of Nacu to create standard error measurements and draw conclusions. By utilizing replicate samples, this would allow the ordinary artisan to have higher confidence in the accuracy of results, provided that the replicates produced similar results, or could indicate a contamination or methodology error in a replicate if the results between replicates were different, providing guidance for next experimental steps. Utilizing standard error measurements would provide a level of confidence for the results and conclusions drawn overall, and would precisely quantify variation between replicate samples. There would be a reasonable expectation of success because the amplification and sequencing methods of Saffroy, in view of Carson, in view of Bramlett, and in view of Salzman itself would not be changing, the method would just be repeated several times for each replicate and there would be additional data analysis steps. Additionally, calculating standard error is a well-known practice in the art, as evidenced by Nacu.
Therefore, claims 18-19 are prima facie obvious over Saffroy, in view of Carson, in view of Bramlett, in view of Salzman, and further in view of Nacu.
Claims 26-28 are rejected under 35 U.S.C. 103 as being unpatentable over Saffroy et al. (US 2016/0319365 A1), in view of Carson et al. (US 2010/0086918 A1), in view of Bramlett et al. (US 2016/0194694 A1), in view of Salzman et al. (PLoS Biology, 2011; cited in Applicant’s IDS), and further in view of Odegaard et al. (Clinical Cancer Research, 2018).
Regarding claim 26, Saffroy, in view of Carson, in view of Bramlett, and in view of Salzman teach the method of claim 1, as described above. However, none of these references discuss samples with specific allele fractions as related to gene fusion data.
It is noted that instant claim 26 still does not require the determination of the allelic fraction in the replicate samples, but simply that replicate samples with a particular allelic fraction(s) be used. See “Claim Interpretation” above.
Odegaard teaches the use of sequencing methods to examine circulating cell-free tumor DNA (Abstract). The reference teaches that sequencing methods are able to detect genetic fusions (page 3539, column 1, para. 1). Fusion events were detected via an analysis of sequencing data (page 3541, column 1, para. 1). The reference teaches that their assay design can detect fusions at allele fractions of 0.04% (page 3542, column 2, para. 1), though fractions of slightly higher than 0.2% were used, as all fusions present in a sample were detected at this level (page 3543, column 1, para. 2 and see Figure 3A). Controls were also used that had allelic fractions of 1-2% (page 3544, column 1, para. 1). It is noted that the sequencing methods used by Odegaard were paired-end, standard NGS practices that utilized well-known products and kits (e.g. see the Agilent and Illumina products mentioned on page 3540, column 2, para. 2). Odegaard also teaches the use of replicates for the same allele frequencies in their validation data (page 3541, column 2, para. 2) and alludes to the use of replicate samples in Figure 2B.
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 Odegaard to use samples with an allelic fraction of slightly more than 0.2% or greater in the method of Saffroy, in view of Carson, in view of Bramlett, and in view of Salzman. Odegaard teaches that sequencing of fusion samples with said allelic fractions is possible, and that data can be effectively examined as it relates to disease. At just above 0.2% allelic fraction and higher, all fusions present in the sample were able to be detected, and so this would motivate the ordinary artisan to use allelic fractions of at least these percentages so as to ensure that all genetic fusions present in a test sample are counted. This would increase the accuracy of the data set and any results/conclusions drawn. Additionally, the range for allelic fractions described by Odegaard overlaps with several of the values in instant claim 26, namely 1%, 0.5%, and 0.25%. MPEP 2144.05 I states, “In the case where the claimed ranges "overlap or lie inside ranges disclosed by the prior art" a prima facie case of obviousness exists. In re Wertheim, 541 F.2d 257, 191 USPQ 90 (CCPA 1976); In re Woodruff, 919 F.2d 1575, 16 USPQ2d 1934 (Fed. Cir. 1990).” Additionally, MPEP 2144.05 II (A) states, “Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. "[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955).” Applicant has not provided evidence that the claimed allelic fractions (which amount to allelic concentrations) are critical or unexpected. As this combination amounts to simply using a particular type of sample and does not change any of the method steps/manipulations of Saffroy, in view of Carson, and in view of Bramlett, there would be a reasonable expectation of success. It is noted that this reasoning would apply to each replicate sample used in the method of Saffroy, in view of Carson, in view of Bramlett, and in view of Salzman.
Thus, claim 26 is prima facie obvious over Saffroy, in view of Carson, in view of Bramlett, in view of Salzman, and further in view of Odegaard.
Regarding claim 27, as noted above, Odegaard teaches a wide range of potential allelic frequencies that were examined. It would be prima facie obvious that for the replicate samples of Saffroy, in view of Carson, in view of Bramlett, and in view of Salzman, different allelic fraction could be used for each replicate in order to compare detection rates given said different fractions. This would aid in determining if, for a particular disease or cancer, if there are certain fractions at which detection is very low or not possible, and conversely, allelic fractions at which detection is essentially guaranteed. This can be compared to disease stages for patients and then used to infer prognostic data. Additionally, using different allelic fractions would allow for the comparison of different primer designs, amplification methods, and sequencing methods for particular gene fusions, to determine if changes in protocol result in better or worse gene fusion detection. This could then aid in optimizing non-invasive liquid biopsy diagnostic methods for particular diseases/cancers. There would be a reasonable expectation of success as this would simply involve using multiple replicate samples with allelic fraction within the range described above in Saffroy, in view of Carson, in view of Bramlett, in view of Salzman, and further in view of Odegaard.
Thus, claim 27 is prima facie obvious over Saffroy, in view of Carson, in view of Bramlett, in view of Salzman, and further in view of Odegaard.
Regarding claim 28, as noted above, Odegaard teaches making multiple replicates at a specific allele frequency (page 3541, column 2, para. 2). It would be prima facie obvious to make each replicate sample in the method of Saffroy, in view of Carson, in view of Bramlett, in view of Salzman, and further in view of Odegaard have the same allelic fraction so that each replicate would act as a check on the others. If each replicate had the same allelic fraction, then each replicate should show the same or very similar results to one another. If this was not the case, it could indicate an unaccounted for error source or issue with reagents/protocols. This check would be valuable because it would increase the accuracy and confidence of downstream analyses. There would be a reasonable expectation of success as this would simply involve using multiple replicate samples with allelic fraction within the range described above in Saffroy, in view of Carson, in view of Bramlett, in view of Salzman, and further in view of Odegaard.
Thus, claim 28 is prima facie obvious over Saffroy, in view of Carson, in view of Bramlett, in view of Salzman, and further in view of Odegaard.
Claim 31 is rejected under 35 U.S.C. 103 as being unpatentable over Saffroy et al. (US 2016/0319365 A1), in view of Carson et al. (US 2010/0086918 A1), in view of Bramlett et al. (US 2016/0194694 A1), in view of Salzman et al. (PLoS Biology, 2011; cited in Applicant’s IDS), and further in view of Vargas et al. (PLoS ONE, 2016).
Regarding claim 31, Saffroy, in view of Carson, in view of Bramlett, and in view of Salzman teach the method of claim 1, as described above. However, none of these references discuss selective PCR.
Vargas teaches a method of selective, non-symmetric PCR utilizing SuperSelective primers that enable the detection of rare DNA fragments in blood samples, even in the presence of large amounts of wild-type DNA (Abstract). This PCR can also be performed in multiplex (Abstract). Figure 1B shows that the use of these primers results in earlier detection of mutants compared to wild-type samples alone. Similar results are shown in Figure 8 for multiplex samples. Vargas concludes that their methods are able to provide low-cost, sensitive, and flexible assays that are able to detect small amounts of mutation in a sample, particularly to compare with reference wild-type abundance (Pages 22-23, “Future directions”).
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 guidance provided by Vargas to utilize the selective PCR taught by the reference in the method of Saffroy, in view of Carson, in view of Bramlett, and in view of Salzman. This combination of references already utilizes multiplex PCR, and is intended to examine gene fusions, which as evidenced by Salzman, can sometimes be difficult to detect in samples, and replicates are needed to determine if a patient/test sample is truly fusion-positive. As gene fusions may be present in small numbers compared to wild-type sequences, and thus may be difficult to detect, the methods of Vargas, which focus on amplification of low-frequency mutations without also amplifying similar non-target sequence, would be appealing to the ordinary artisan in the context of the methods of Saffroy, in view of Carson, in view of Bramlett, and in view of Salzman. As Vargas teaches that their methods are successfully in a multiplex content, the design of their primers is clearly laid out (see Figure 1A), and methods of PCR amplification are well-known in the art (see Saffroy, Carson, Bramlett, and Salzman), there would be a reasonable expectation of success.
Thus, claim 31 is prima facie obvious over Saffroy, in view of Carson, in view of Bramlett, in view of Salzman, and further in view of Vargas.
Conclusion
No claims are currently allowable.
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/FRANCESCA FILIPPA GIAMMONA/Examiner, Art Unit 1681
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