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 19-22) and Species B (claims 30-31) in the reply filed on 12/10/2025 is acknowledged.
Claims 15-18 and 23-31 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim.
Claim Objections
Claim 19 is objected to because of the following informalities: in line 1 of step 2), “substituting Ctx-LH in and CTx-HT obtained in step 1)” should read “substituting the values for Ctx-LH and CTx-HT obtained in step 1)” for better clarity. It is noted that “the values” should only be used if Applicant moves the wherein clause in the claim as described below. For clarity, it is also recommended that the wherein clause below step 2) be placed below step 1), as this clause explains the variables first described in step 1). If this change is made, line 1 of step 2) should also read “to obtain ΔCtx for the target gene” rather than “to obtain ΔCtx of a target gene.” Additionally, in line 4 of step 3) “logarithm relationship” should read “logarithmic relationship.” 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 19-22 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 19 is rejected because the metes and bounds of the claim are unclear. In step 3), multiple phrases are unclear. Firstly, this step requires the use of “fragments of different DNA methylation levels,” and the amplification of said fragments for the target gene. It is unclear how DNA methylation levels, which are generally single values or a range of values, could be fragmented, and as DNA methylation levels are not DNA themselves, how they could be amplified. It will be interpreted as though fragments of the target gene must be amplified, where either multiple copies of a single target gene with different DNA methylation levels are used, or where “the target gene” can include multiple individual genes with different DNA methylation levels. This latter interpretation is supported by claim 20, which states that the “target gene” may include multiple genes, as well as para. 26 of the instant specification, which states “When the target gene includes two or more target genes…” It is noted that both natural and unnatural methylation (e.g. bisulfite treatment) is encompassed by this step, as Applicant does not specify the type of methylated DNA to be used.
Step 3) of this claim is further unclear because line 2 states that the amplification creates “resulting PCR data,” but the amplification of the fragments is not specified to be PCR. It is recommended that Applicant specifically state that PCR is to be used during amplification in this step.
Further regarding this step, lines 2-3 state “subjecting resulting PCR data to the operations in step 1) and step 2)” (emphasis added). A value for ΔCtx must be obtained through this subjecting, and so it seems the fragments would need to undergo additional amplification at high- and low- denaturation temperatures. It is unknown if these amplification steps are intended to be the “PCR data” recited earlier in the step, or if additional amplification other than the initial fragment amplification is being recited (i.e. amplifying the target gene three times in step 3)). As steps 1) and 2) already include multiple amplification steps, it is unknown if these amplification steps would be considered part of “the operations” of steps 1) and 2). “The operations” does not have a clear scope, and so may include any or all of each of steps 1) and 2). Step 3) will be interpreted as though either two or three amplification steps may be used (where high- and low- denaturation temperature amplification must be used regardless).
In step 4) of claim 19, it is also unclear how comparing ΔCtx to ΔCtx’ would allow one to obtain a proportion of methylated cytosine in the target gene, as ΔCtx and ΔCtx’ are not measures of methylation. Additionally, the measurements initially made in steps 1) ad 2) have no particular requirements with regards to methylation. Finally, the language “with ΔCtx’ in the reference curve” is unclear, because ΔCtx’ values are single numbers, and those values would remain the same on or off the axis of the reference curve.
Claims 20-22 are rejected due to their dependence on rejected claim 19.
Claim Interpretation
It is noted that in claim 19, the primers and probes of claim 15 are used “to prepare a reaction system for qPCR detection,” and then only the use of “high-temperature denaturation amplification” and “low-temperature denaturation amplification” are described. The amplification reactions are not required to be performed with the stated primers and probes or within the stated reaction system, and so will not be considered to be limited to qPCR amplification. Prior art will therefore read on the claimed primers and probes if primers and probes are generally used in an amplification/detection system. The amplification of step 3) will also be similarly interpreted as not requiring qPCR, particularly as the “PCR data” generated does not have to be qPCR data.
The term “reference curve” is not specifically defined in the instant specification. Para. 70 of the instant specification states that “ΔCtx of a target gene is compared with ΔCtx' in the reference curve to obtain a proportion of methylated cytosine in the target gene, such as to determine a DNA methylation level of the target gene. In this way, a DNA methylation level of each target gene can be quantitatively evaluated through the reference curve.” However, in paras. 93 and 96, the reference curve appears to be drawn relative to proportions of different methylation levels. Thus, any curve which includes multiple ΔCt’ values, where said ΔCt’ values can be used in conjunction with ΔCt values to determine methylation levels of a target, will be considered to meet this limitation.
As noted above in the 35 USC 112(b) Rejection section, claim 19 contains several indefiniteness issues. Considering these indefiniteness issues and the broadest reasonable interpretation of the claim, the claim will generally be interpreted as: performing amplification at high- and low- denaturation temperatures on an initial target, and finding a ΔCt value based on these amplifications according to equation 1. Then, this is repeated for a fragmented target, resulting in a ΔCt’ value, wherein the fragmented target in the second sample differs from the first in a manner related to DNA methylation and contains multiple methylation levels. A reference curve as described above, including multiple ΔCt’ values, is then made. The initial ΔCt value is then used with one or more ΔCt’ values to determine the methylation level in the initial target sample.
Claims 21-22 recite particular reaction conditions for the high- and low- temperature denaturation amplification reactions. 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)…Similarly, a prima facie case of obviousness exists where the claimed ranges or amounts do not overlap with the prior art but are merely close. Titanium Metals Corp. of America v. Banner, 778 F.2d 775, 783, 227 USPQ 773, 779 (Fed. Cir. 1985)” 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) (Claimed process which was performed at a temperature between 40°C and 80°C and an acid concentration between 25% and 70% was held to be prima facie obvious over a reference process which differed from the claims only in that the reference process was performed at a temperature of 100°C and an acid concentration of 10%.).”
In the instant specification, para. 22 states “Reaction conditions for the high-temperature denaturation amplification vary depending on a gene sequence of a target region…” with similar language being used in para. 23 for the low-temperature denaturation amplification. Similar language is used in paras. 82-83. Though the working examples utilize the claimed reaction conditions (e.g. paras. 115-116), these conditions are never described as critical or producing unexpected results. Additionally, alternative PCR conditions are also described in the working examples (see the SDC2 amplification in para. 176 for instance). Therefore, the reaction conditions described by claims 21 and 22 will be considered routine experimentation, and prior art that teaches reaction conditions with the values of/within the ranges described by the claims or values/ranges close to those claimed, will be considered to read on the reaction conditions of the instant claims.
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.
Claims 19-20 are rejected under 35 U.S.C. 103 as being unpatentable over Milbury et al. (JMD, 2011), in view of Guo et al. (US 2018/0148776 A1), and in view of Mauger et al. (Epigenomics, 2018).
Milbury teaches the use of conventional PCR and COLD-PCR on DNA from cancer cells (Abstract). The gene TP53 (also known as P53) was examined (Table 1, page 221, column 2, para. 3, and Table 2, for example; instant claim 20) in colorectal and glioma tumor tissue (page 221, column 2, para. 4), as well as genomic DNA from cancer cell-lines and control genomic DNA (page 221, column 2, para. 3). Wild-type and mutation samples were compared in both PCR conditions using fluorescence difference curves, where multiple curves were made based on serial dilutions of the cancer cell-lines in wild-type DNA (page 225, column 1, para. 3 and Figure 1, for example). Generally, the higher the mutation percentage present in a sample, the higher the fluorescence value became, and COLD-PCR seemed to show even greater fluorescence compared to conventional PCR for at least one exon (Figure 1), The primers used for PCR are shown in Table 2. It is noted that although genomic DNA is used, the amplification performed is specific for TP53, and before conventional and COLD-PCR were performed, multiplex amplification occurred to specifically create amplicons containing exons and splice regions of TP53. Thus, this initial amplification produced fragmented DNA that was further amplified (page 222, column 1, para. 4).
However, Milbury does not teach the specific Ct value methods of the instant invention, nor the use of probes. The reference also does not specifically subtract high-denaturation temperature Ct values from low-denaturation temperature Ct vales. The reference also does not discuss methylation in depth, though it does state, “The majority of human TP53 mutations are Tm-reducing, a bias which putatively reflects the methylation of 5’-CpG-3’ dinucleotides,” (page 228, column 2, para. 2), indicating that an increase in TP53 methylation can be associated with TP53 mutation, and therefore cancer risk.
Guo teaches methods for determining a methylation profile in a DNA sample in the context of cancer (Abstract). The general method involves use of primers and probes for amplification and detection, as well as materials for detecting methylation sites (including digestion materials), where fluorescent probe signals are used to determine a genomic DNA methylation profile (para. 5). qPCR can specifically be used (para. 31). Determining a methylation status can comprise determining the proportion of nucleotide positions that are methylated in the genomic region (para. 33). Multiplex amplification is shown to work with these methods (para. 45 and Figure 2). The general methylation methods are shown in para. 54 and Figure 15. Unmethylated DNA is digested and will not be further amplified in a particular sample, while methylated DNA will be amplified. Para. 58 and Figure 19 then explain how this is used to calculate methylation percentage. ΔCt values are calculated for digested and undigested samples (see para. 134), and then these values are subtracted from one another and manipulated to obtain a methylation percentage. The initial ΔCt values are based on the Ct of an individual marker as compared to a control (para. 48). Figure 24 shows examples of standard curves with Cq values for a given starting quantity of a sample.
Mauger teaches the detection of DNA methylation patterns with COLD-PCR, and compares the results of said analyses with those of qPCR (Abstract). Figure 2 shows the creation of standard curves for DNA methylation based on the proportion of methylated DNA in a sample, and compares multiple sites across two genes for qPCR and COLD-PCR. Figure 3 also shows percent methylation for qPCR, COLD-PCR (using the standard curves, see para. 2 on page 530), and quantification methods for COLD-PCR. Mauger states, “DNA methylation has been proposed as an alternative molecular alteration that might be used for the detection and follow-up of cancerous lesions…The frequent and localized presence of aberrant DNA hypermethylation patterns in the promoter regions of cancer-related genes bears therefore great promise for personalized medicine of cancer or other diseases,” (page 526, para. 1).
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 combine the teachings of Milbury, Guo, and Mauger to arrive at the invention of claim 1. Firstly, Guo teaches the use of probes to determine fluorescence information during qPCR – this would make fluorescence detection (e.g. via Ct values) simpler as compared to the methods used by Milbury alone, where samples undergo amplification and then are further processed to obtain fluorescence information (page 225, column 1, para. 3). Including probes within amplification reactions would shorten protocol time, as fluorescence detection would be happening during the amplification reaction and not in a later step. Additionally, Milbury includes conventional PCR mainly as a means for comparison with COLD-PCR (see “Conventional PCR-HRM was performed in parallel for comparison with COLD-PCR-HRM” on page 225, column 1, para. 3 and “COLD-PCR-HRM analysis results in an approximate three-fold improvement in detection sensitivity over HRM analysis of conventional PCR amplicons…” on page 225, column 2, para. 2, for example). Thus, the conventional PCR is acting as a control for COLD-PCR by providing a well-known, baseline means for comparison. Mauger provides support for this line of thinking by specifically comparing qPCR and COLD-PCR methods, and provides reason to examine methylation in cancer specific genes with these amplification methods. Milbury also specifically discusses that methylation of TP53 is likely related to cancer, further connecting the teachings of these references.
Relating this to Guo, as shown in Figure 19 of the reference, to obtain methylation percentages for samples, average Ct values for a target are compared to those of a control to obtain a first ΔCt value. Then, ΔCt values for digested and undigested samples are compared to determine the percentage of methylation in a digested sample (where the methylation is represented as a percentage of the value obtained in the undigested sample). Mauger utilizes pyrosequencing to determine methylation levels (page 528, para. 2), but by simply utilizing Ct and ΔCt values, this would prevent the need for providing sequencing equipment and taking the additional time to perform said sequencing and analyze results. As the ΔCt values are based on Ct values gathered during amplification, these provide a faster way to obtain methylation values, and can be obtained using the probes described in the paragraph above.
Thus, the ordinary artisan would be motivated to alter the calculations of Guo slightly to compare qPCR and COLD-PCR across target and control samples to find the methylation levels in the target sample, as well as to better evaluate any mutations in said target sample. Both Milbury and Mauger note that COLD-PCR increases the sensitivity of mutation detection (Milbury page 231, column 1, para. 3 and Mauger page 534, “Conclusion”), and Milbury already teaches the use of wild-type control samples. Given that cancer samples already have altered methylation levels compared to non-cancerous samples, this would entail finding a first ΔCt value by subtracting the Ct values of the qPCR from those of the COLD-PCR (as the qPCR is acting as the control, similar to POLR2A in Figure 19A of Guo), then finding ΔΔCt by subtracting the control wild-type samples from the target cancer samples. Then, the manipulations of Guo would proceed as normal, and the methylation value reached at the end would be a proportion of methylation in the cancer samples relative to a baseline, wild-type level. These calculations would be valuable, as this methylation level comparison for TP53 could then be used as non-invasive diagnostic criteria for the cancers examined by Milbury (colorectal cancer, glioma, etc.), as Milbury teaches detection of this gene in conjunction with these diseases. This method would also be valuable as all the detection would occur in conjunction with the PCR methods, and so additional sample manipulation would not be required, and all downstream methods would be data analysis and manipulation, saving time and resources.
Finally, as to the reference curve stated in the claim, Guo creates standard curves for multiple Cq values relative to a starting amount of a sample (see Figure 24). As the mathematical manipulations of Guo involve using multiple Ct values to obtain ΔCt values, and Milbury teaches that their PCR for tumors involves different amounts of starting sample and additional serial dilutions (page 222, column 1, para. 3), and the cancer cell-line analyses used also included serial dilutions (page 222, column 2, para. 1, see also page 223, column 1, para. 2 and page 225, column 1, para. 2), the ordinary artisan would see the value of creating such a standard curve for the analysis of the tumor samples. Such a standard curve can provide a check on the method (i.e. ensuring that the values obtained generally follow a typical standard curve), as well as allowing for checks to the method for future repetitions. This would alert the ordinary artisan to potential issues relatively early on during amplification, which would limit experimental waste of both time and resources.
As for a reasonable expectation of success for this combination of references, the changes amount to adding probes to amplification reactions to obtain Ct values (which is done by Guo, providing evidence such a reaction mixture is possible), and then manipulating the Ct values and resulting data in particular ways as described by Guo. As such calculations are relatively simple and well-known, as evidenced by Guo, they would be possible for the ordinary artisan to perform.
Thus, claims 19-20 are prima facie obvious over Milbury, in view of Guo, and in view of Mauger.
Claims 21-22 are rejected under 35 U.S.C. 103 as being unpatentable over Milbury et al. (JMD, 2011), in view of Guo et al. (US 2018/0148776 A1), in view of Mauger et al. (Epigenomics, 2018), and further in view of Li et al. (Nature Medicine, 2008) and Al-Kashwan et al. (BMC Research Notes, 2012).
Regarding claims 21-22, in the PCR methods utilized by Milbury, Table 3 shows the reaction conditions for each. It is noted that reference is also made to values stated in Table 2. In comparing the conventional PCR reaction conditions of Milbury to the high-denaturation temperature conditions of instant claims 21-22, there is as follows:
Initial denaturation
Cycling denaturation
Cycling annealing
Cycling extension
Number of Cycles
Claimed Reaction Conditions
95°C for 5 minutes
94°C for 5 seconds
60-62°C for 15 seconds
72°C for 30 seconds
45
Milbury Reaction Conditions
98°C for 30 seconds
98°C for 10 seconds
57-70°C for 30 seconds
72°C for 10 seconds
35
As noted above in the “Claim Interpretation” section, the claimed PCR reaction conditions are noted to depend on the specific target region used, and are overall not considered critical or to produce unexpected results. Therefore, prior art that teaches the same conditions, or conditions close to, those claimed, will be considered to read on the instant claims. In comparing the PCR values in the above table, conditions between the claimed invention and Milbury are considered to be the same or close to one another for all except the bolded portion.
In comparing the COLD-PCR reaction conditions of Milbury to the low-denaturation temperature conditions of instant claims 21-22, see the following table. It is noted that the Stage 2 and Stage 3 cycling of Milbury is recited, as the Stage 1 cycling does not involve low-temperature denaturation. Such additional cycling is not prohibited by the instant claims (i.e. the method of claim 19 comprises the listed steps).
Cycling denaturation
Cycling annealing
Cycling extension
Number of Cycles
Claimed Low-Temperature Denaturation Conditions
85-88°C for 15 seconds
60-62°C for 15 seconds
72°C for 30 seconds
18
Milbury Low-Temperature Denaturation Conditions
85.9-93.5°C for 10 seconds
57-70°C for 20 seconds
72°C for 10 seconds
20
Claimed Secondary Reaction Conditions
94°C for 5 seconds
60-62°C for 15 seconds
72°C for 30 seconds
27
Milbury Secondary Reaction Conditions
98°C for 10 seconds
57-70°C for 20 seconds
72°C for 10 seconds
10
Similar to the scenario for the high-temperature denaturation, as recited in the “Claim Interpretation” section above, prior art that teaches the same conditions, or conditions close to, those claimed, will be considered to read on the instant claims. In comparing the PCR values in the above table, conditions between the claimed invention and Milbury are considered to be the same or close to one another for all except the bolded portion.
Al-Kashwan teaches studying multiple mutations in TP53 in relation to cancer (Abstract). Target sites were each amplified via PCR, where for each site, an initial denaturation step at 95°C for 5 minutes was performed (page 3, column 1, para. 2).
Li teaches the use of COLD-PCR for analysis of target sequences, including TP53 (Abstract). On page 584, column 1, the COLD-PCR conditions used are listed. For TP53, during both full and fast COLD-PCR cycling, a series of 25 cycles was performed with typical denaturation temperatures, while 30 cycles were performed with lower denaturation temperatures (see the final paragraphs of both the “Full-COLD-PCR” and “Fast-COLD-PCR” sections).
Though the general reaction conditions of Al-Kashwan and Li are not the exact same as those of Milbury or the instant invention, these references show that a range of PCR and COLD-PCR reaction conditions may be used to amplify a single target gene. Al-Kashwan and Li focus on the same gene as in Milbury (TP53), and provide values for the conventional PCR initial denaturation time (in the case of Al-Kashwan) and the COLD-PCR numbers of cycles (in the case of Li) the same as or close to those of the instant invention. These denaturation time and cycle number values of Al-Kashwan and Li, respectively, therefore render those of the instant invention prima facie obvious for the reasons stated above in the “Claim Interpretation” section and in paras. 33 and 35 of this rejection.
Thus, claims 21-22 are prima facie obvious over Milbury, in view of Guo, in view of Mauger, and further in view of Al-Kashwan and Li.
Conclusion
No claims are currently allowable.
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/FRANCESCA FILIPPA GIAMMONA/Examiner, Art Unit 1681