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 elected, without traverse, a specific combination of HPV and a specific amplicon in the reply filed on 03/31/2025.
Upon further reconsideration, the requirement for a specific amplicon and combination of HPV serotypes is withdrawn.
Status of Claims
Applicant' s amendment filed 11/06/2025 is acknowledged. Claims 27-34 have been added. Claims 1-18 and 20-26 have been cancelled. Claims 27-34 are pending in the instant application and claims 27-34 are the subject of this final office action.
All of the amendments and arguments have been reviewed and considered. Any rejections or objections not reiterated herein have been withdrawn in light of amendments to the claims or as discussed in this office action.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Previous Rejection
Status of Prior Rejections/Objections:
The 112(b) rejection to claims 16-18 and (now, respectively, 27 and 30-32) and 20 (now 32) are withdrawn in view of the amendments to the claims.
The prior art rejection(s) under 35 USC 103 directed to claim(s) claims 16-18 and 20 (now, respectively, 27 and 30-32) as being unpatentable over Eloit in view of Kassambara are maintained and modified as necessitated by amendments.
The objection to the specification regarding the hyperlink is withdrawn.
The claim objection to claim 20 is withdrawn.
Reference Notes
An updated copy of Kassambara has been provided given the previous quality of automated the color-to-greyscale conversion.
New Ground(s) of Rejections
The new ground(s) of rejections were necessitated by applicant’s amendment of the claims.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claim Objections
Claim 32 is objected to because of the following informalities:
Claim 32:
The claim has extra commas after the blocks corresponding to i=4 and i=5.
The claim is missing a comma after each of the blocks corresponding to i=7, i=8, and i = 23.
Appropriate correction is required.
Claim Interpretation
In evaluating the patentability of the claims presented in this application, claim terms have been given their broadest reasonable interpretation (BRI) consistent with the specification, as understood by one of ordinary skill in the art, as outlined in MPEP 2111.
Regarding claims 27, 32, and 33, the instant specification defines a “splice junction i” for each of the HPV types in Tables 1 and 2A according to “SD” and “SA” designations (i.e., i = 1 is defined as SD3-SA4), which are defined as a genomic locations for each of the HPV types in Fig. 2 (pg. 56, lines 9-10).
Thus, it is interpreted that a sum of the beta-scaled X values for each of the expression levels of splice junctions i = 1 through i = n would consist of the continuous set of values beta_1*X_1,j + beta_2*X_2,j + … + beta_n*X_n,j, wherein each i in the set {1, 2, … , n} corresponds to the defined splice junction.
It is further noted that where data is not collected/provided, it would be understood by the artisan that the X values being zero would cause those values to “drop out”.
Claim Rejections - 35 USC § 103
Claims 27-34 are rejected under 35 U.S.C. 103 as being unpatentable over Eloit (WO 2016/005905 A2; published 01/14/2016; as cited in the IDS dated 09/19/2021) and Kassambara (Kassambara. Logistic Regression Essentials in R [online]. 2018 [retrieved on 2025 May 21]. Retrieved from the Internet: https://www.sthda.com/english/articles/36-classification-methods-essentials/151-logistic-regression-essentials-in-r/).
Regarding claims 27-30, Eloit teaches a method comprising:
Extraction of viral RNA from a biological sample (pg. 107, line 4)
Reverse transcription of the RNAs into cDNA (pg. 107, line 5)
Amplification of the cDNA by multiplex PCR to generate a DNA sequence database, wherein the multiplex amplification is performed with HPV-specific primer pairs (pg. 107, lines 6-8)
High throughput sequencing of the DNA library and generation of sequencing reads (pg. 107, line 12) and aligning reads with the sequences of the HPV genomes present in the database (pg. 107, line 14) [i.e., quantifying the expression level of each cDNA amplicon in the “DNA sequence database”, i.e., a database of amplicon sequences].
Computing a score R based on the number of reads generated by at least 2 pairs of primers (pg. 107, line 16)
Eloit teaches extending the method to the entire alpha-HPV group, i.e., HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 73 and 82 (instant claim 28), based on the transcription map described in Fig. 7 using the primers of Table 14 (pg. 82, lines 27-28; Table 14).
Appendix A maps a subset of the primers of Table 14 using the annotations on pg. 44-45 (necessitated by OCR limitations) and instant Table 2A to identify primer pairs in Table 14 that correspond to each HPV type.
As shown in Appendix B, the primers taught in the instant disclosure for the first through thirteenth subsets (instant primers SEQ ID NO: 1-430 corresponding to instant claimed amplicons SEQ ID NO: 1501-1715; see instant Table 2A) correspond to Eloit primer SEQ IDs shown in Appendix A taught for amplification of the splice junctions.
Thus, the artisan would understand that the identical forward and reverse primers of Eloit would inherently produce the same amplicon as the identical instant forward and reverse primers given the same template. Eloit therefore inherently teaches the at least 10 pairs of primers capable of producing the amplicons in the ranges of the claim (instant claims 29 and 30).
Eloit teaches that low-grade intraepithelial lesions are a site of productive viral replication (pg. 2, line 20), and that R scores based on a ratio of [HPV genes] E6 and/or E7 and/or E2 and/or L1 and/or L2 generated high score values associated that should be associated with non-or lowly-productive HPV cycles typical of transformed cells (pg. 65, lines 32-34) [i.e., high grade intraepithelial lesions, HSIL; see pg. 77, lines 1-2 and line 11].
Thus, Eloit teaches a method to discriminate high risk lesions from low risk lesions from the RNA of a biological sample and use of parameters multiplied by the expression of HPV genes to generate a score to do so.
Eloit further teaches characterizing and counting reads associated with specific splice junctions to confirm the detect of transcripts over possible artefacts introduced by residual HPV DNA (pg. 65, lines, 19-21).
Eloit fails to explicitly teach applying a logistic regression and the use of a p-value greater than 0.5 to indicate the presence of an HSIL in the biological sample.
Kassambara rectifies this by teaching a logistic regression used to predict the class of individuals based on one or multiple predictor variables, wherein a variable can have only two possible values, including diseased or non-diseased (para 1). Kassambara teaches that for when you have multiple predictor variables, logistic function looks like: log[p/(1-p)] = b0 + b1*x1 + b2*x2 + ... + bn*xn (Logistic function, para 1 and 4). Kassambara further teaches that the quantity log[p/(1-p)] is called logit (Logistic function, para 5).
Further, the artisan would understand that the notation for sigma is a summation equivalent to that represented with to the groupings with b1*x1 + b2*x2 + … + bn*xn, therefore would understand that the following was equivalent:
l
o
g
i
t
p
=
b
0
+
∑
i
=
1
n
(
b
i
x
i
)
Kassambara teaches that by default, the threshold probability when the category flips from one to the other is set to p=0.5 (para 3).
Kassambara teaches including all predictor variables available in the data set (Multiple logistic regression, para 2) and removing highly corelated predictors to minimize overfitting (Preparing the data, para 1).
Kassambara teaches modeling disease (pg. 1, para 1; pg. 2, “Preparing the Data” through pg. 6, “Discussion”).
Kassambara further teaches that logistic regression model output is very easy to interpret compared to other classification methods and because of its simplicity it is less prone to overfitting than flexible methods such as decision trees (Discussion, para 1).
Therefore, it would have been obvious to one of ordinary skill in the art at the time of filing to replace the method of calculating and R score with ratios of Eloit with the multivariate logistic regression of Kassambara that has a default threshold of p=0.5, wherein it further would have been obvious to utilize the expression of spliced transcripts of Eloit. The artisan would have been motivated to utilize the logistic regression because of the ease of interpretation and the model’s lower chances of overfitting data, as taught by Kassambara, and in further view of Eloit’s use of weighting of expression data for discriminating lesion grades [i.e., that an artisan would recognize are analogous to the weights of a logistic regression]. The artisan would have been motivated to utilize spliced transcripts in view of the teaching of Eloit that the spliced junctions are able to discriminate between [RNA] transcripts and artefacts from contaminating DNA. There would have been a strong expectation of success as these are known techniques with predictable results.
It is noted that in the previous rejection of claim 17 (now claim 30), a count of the putative primer pairs was made according to Fig. 7, wherein an obvious rejection was made according to nomenclature. Table 13 numbers HPV33 pairs at 13, as was referenced in the previous rejection; said difference, when forward and reverse primers are shared amongst pairs (i.e., SD1 pairs share the SD1 forward primer) and the method utilizes a multiplex amplification is a matter of nomenclature.
Regarding claim 31, in the method of Eloit in view of Kassambara, Eloit teaches additional primers pairs (Tables 10 and 14) defined to amplify the boundaries at the 5'-SD-genomic [splice donor spanning genomic] and 5’-genomic-SA-3' positions [genomic spanning splice acceptor] (suffix 'unsp_') [i.e., “unsplice junctions”] to allow for a better quantitative monitoring of concomitant spliced and/or genomic/unspliced transcription events and refine if necessary the description of transcripts equilibrium in the course of HPV infection (pg. 76, para 1).
As cited above, Eloit teaches extending the method to the primers of Tables 14 (pg. 82, lines 27-28), which comprise said unspliced regions.
Regarding claim 32, in the method of Eloit in view of Kassambara, Eloit teaches the pairs of primers above.
Applicant was requested to make a showing of which of the instant amplicons would differ, if any, from those that would be generated from the primers of Eloit in Table 14 given the identity of all sequences checked to those of the primers in instant Table 2A in the previous rejection. No deficiency has specifically been identified.
Appendices A and B elaborate on the previous rejection according to Table 14, further demonstrating that the primers of Eloit produce amplicons for splice junctions i = 1 through i = 9, i = 11, i = 12, i = 15 through i = i = 24.
In addition, Table 14 teaches:
18_sp_3165_3465 (SEQ ID NO: 477 and 478), corresponding to i = 10 (SD4-SA6) for HPV18
18_sp_3786_5613 (SEQ ID NO: 567 and 568), corresponding to i = 13 (SD6-SA9) for HPV18
45_sp_3750_5608 (SEQ ID NO: 1011 and 1012), corresponding to i = 13 (SD6-SA9) for HPV45
18_sp_929_3465 (SEQ ID NO: 531 and 532), corresponding to i = 25 (SD2-SA8) for HPV18
18_sp_3786_5776 3465 (SEQ ID NO: 557 and 558), corresponding to i = 14 (SD6-SA10) for HPV18
45_sp_929_3423 (SEQ ID NO: 943 and 944), corresponding to i = 25 (SD2-SA8) for HPV45
Thus, Table 14 teaches primer sets capable of producing the claimed amplicons i = 1 through i = 25 according to the annotations of instant Table 2A.
Additionally, Eloit further teaches that primers encompassed by the invention are not limited to the sequences defined in the primers depicted below but they can comprise extra bases at the 5' end, for example from 1 to 5 extra bases as extension corresponding to sequences of the corresponding HPVs E6 or E7 and that primers shall be understood as embracing shorter sequences of at least 12, 15, 20 or 25 consecutive bases of the primers featured below, wherein the primers can be extended or [shifted] from 1 to 15 bases depending on the desired specificity of the PCR amplification step and/or on the specificity of the detection step using standard parameters such as the nucleic acid size and GC contents, stringent hybridization conditions and temperature reactions (pg. 13, para 2). Eloit further teaches that low stringency conditions are used when it is desired to obtain broad positive results on a range of homologous targets whereas high stringency conditions are preferred to obtain positive results only if the specific target nucleic is present in the sample (pg. 107, lines 19-22).
It is noted that the splice donor and acceptor sites of Fig. 7 of Eloit are identical to those of instant Fig. 2, to which Eloit Table 14 primers and instant Table 2A primers are directed.
Thus, even should there be a difference identified, if the primers remain targeted to the same approximate locations given the same targeted splice donor and splice acceptor sites (i.e., splice junctions), it is held that such would not be a patentable difference as it is routine to optimize parameters including the start positions of the primers within the art, as indicated by Eloit, under MPEP 2144.05(II), such that the specified amplicons would be arrived at and/or that the resulting amplicons would be obvious variants of one another (i.e., differencing by a few bases based on a primer optimization). As taught by Eloit, the artisan would be motivated to adjust the length and starting position of primers in order to alter the GC content, nucleic acid size, temperature reactions, etc. to optimize them for low or high stringency conditions in order to optimize for particular sample preferences.
Regarding claim 33, the claimed splice junctions correspond to i=1 (SD3-SA4), i=2 (SD3-SA5), i=3 (SD3-SA6), i=4 (SD1-SA4), i=5 (SD1-SA5), i=6 (SD1-SA6), i=7 (SD1-SA1), i=8 (SD1-SA2), i=9 (SD1-SA3), i=11 (SD5-SA9), i=12 (SD5-SA10), i=15 (SD2-SA4), i=16 (SD2-SA5), i=17 (SD2-SA6), i=18 (SD2-SA9), i=19 (SD2-SA10), and i=25 (SD2-SA8).
The absent i=10, i=13, i=14, and i=20…24 represent, in part, a choice to remove the junctions associated with SD4, SD6 and SA7. Specifically absent associated with these splice sites are i=10 (SD4-SA6), i=13 (SD6-SA9), i=14 (SD6-SA10), i=20 (SD1-SA7), i=21 (SD2-SA7), and i=22 (SD3-SA7).
In the method of Eloit in view of Kassambara, Eloit teaches taking into consideration nearest neighbor splice sites in order to minimize the risks of co-amplifying several spliced isoforms with a given couple of primers (pg. 76, lines 1-2). As discussed above, Eloit teaches optimization of the conditions to maximize specific binding.
Eloit further teaches the 1.4 million individuals are each year at risk of developing HPV induced cancer and HPV vaccines cover only a subset of HPV types and leave unprotected a significant part of the population (pg. 1, para 2). Eloit teaches that cancer biomarkers in HPV-related cancers are greatly needed for better diagnostic of pre-cancer and cancer stages of the disease, prognosis, and therapeutic management (pg. 2, para 1). Eloit teaches that all current molecular tests use molecular techniques based on specific amplification/probes of papillomavirus nucleic acids, wherein they are restricted to some specific HPVs, but fail to test for a general and broad range of HPVs (pg. 3, para 3).
The remaining junctions i=23 (SD3-SA8) and i=24 (SD1-SA8) are not associated with a reduced primer set.
Kassambara rectifies this by teaching that an optimization of logistic regression wherein variables that are not statistically significant in a model should be eliminated because keeping them may contribute to overfitting, wherein this processing may be done automatically using statistically techniques including stepwise regression and penalized regression (pg. 5, Interpretation).
Therefore, while method of Eloit in view of Kassambara does not specifically teach the limited combination of splice junctions of the claim, such a reduction in the variables chosen in the logistic regression would be a matter of routine optimization of the method under both the optimization of amplification conditions, as taught by Eloit, and the logistic regression variables, as taught by Kassambara.
Additionally and/or alternatively, would have been obvious to try such a combination by utilizing the regression optimization functions taught Kassambara to add/remove variables from the limited number of splice junction amplicons taught by Eloit, motivated by the market need of a large population vulnerable to HPV cancers and the need for better diagnostic tools for prognostic and therapeutic management of pre-cancerous and cancerous growths applicable to a broader range of HPVs, as taught by Eloit. There would have been a strong expectation of success given the finite number of permutations of variables and widely used automated algorithms for narrowing them down based on the data (i.e., those taught by Kassambara).
Regarding claim 34, in the method of Eloit in view of Kassambara, teaches that the multiplex amplification is performed according to Example 17.3 (pg. 107, lines 6-7), wherein Example 17.3 teaches that a selection of human transcripts was included in the design for normalization purposes and/or to support/improve a combination of human and/or HPV transcripts being able to discriminate low grade vs. high grade lesions of the cervix (pg. 76, line 10 spanning pg. 77, lines 1-2; see also pg. 81, 17.9 Multiplex amplification and quantification of HPV16 transcripts).
Eloit teaches the primer pairs directed to said transcripts in Table 10 within said Example.
Appendix C maps the instant SEQ ID NO: 1441-1500 corresponding to Eloit SEQ ID NO: 159-218, i.e., the “Human Transcripts” section of Table 10, which consists of 30 pairs.
Response to Arguments
Applicant's arguments filed 11/06/2025 have been fully considered but they are not persuasive.
In response to applicant’s argument that there is no teaching, suggestion, or motivation to combine the references, the examiner recognizes that obviousness may be established by combining or modifying the teachings of the prior art to produce the claimed invention where there is some teaching, suggestion, or motivation to do so found either in the references themselves or in the knowledge generally available to one of ordinary skill in the art. See In re Fine, 837 F.2d 1071, 5 USPQ2d 1596 (Fed. Cir. 1988), In re Jones, 958 F.2d 347, 21 USPQ2d 1941 (Fed. Cir. 1992), and KSR International Co. v. Teleflex, Inc., 550 U.S. 398, 82 USPQ2d 1385 (2007). In this case, it would be obvious to combine Eloit and Kassambara by utilizing the spliced transcripts of Eloit and the logistic regression of Kassambara, motivated by Eloit’s teaching that spliced junctions are able to discriminate between [RNA] transcripts and artefacts from contaminating DNA and Kassambara’s teaching that logistic regressions have easy interpretation and low chances of overfitting data. There would have been a strong expectation of success as these are known techniques with predictable results, wherein both are directed towards predicting diseases.
In response to applicant's argument that the references fail to show certain features of the invention, it is noted that the features upon which applicant relies (i.e., “the extent of correct classification of an HSIL shown by Applicant in the Specification”) are not recited in the rejected claim(s). Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993).
Regarding applicant’s argument that the instant invention provides unexpectedly superior results over the cited references, first, MPEP 716.01(c) makes clear that “[t]he arguments of counsel cannot take the place of evidence in the record” (In re Schulze, 346 F.2d 600, 602, 145 USPQ 716, 718 (CCPA 1965)). Evidence of unexpected results, when timely filed, in an affidavit or declaration will be fully considered.
It is noted that the Response above should not be construed as an invitation to file an after final declaration. See MPEP 715.09.
Second, MPEP 716.02(a)(I) states that when greater than expected results are found (i.e., “an exceedingly high level of accuracy”, Remarks, pg. 13, para 4) applicants must show that the results would be greater than those which would have been expected from the prior art to an unobvious extent, and that the results are of a significant, practical advantage.
Third, unexpected results must be commensurate in scope with claimed invention. See MPEP 716.02(d). The specific claims being addressed by the unexpected results arguments have not been identified, and the data indicated is not commensurate in scope with the independent claim.
Fourth, the artisan would understand that accuracy (e.g., PPV) of classification represents a choice based on trade-offs amongst true positives, true negatives, false positives, and false negatives (e.g., based on the threshold probability chosen). The default threshold (see Kassambara, as cited in the 103 rejection) for logistic regression was chosen; thus, the results are expected.
Further, accuracy cannot effectively be assessed using the “training” data presented in the arguments, not least in view of Kassambara’s teachings on overfitting (as cited in the 103 rejection); rather a showing should be made using “test” data. Indeed, contrary to arguments, the PPV of the 13 test HSILs was a lower 81.8% (instant specification, pg. 195, para 3; Table 7).
The applicant noted Perot (Pérot P, et al. Broad-Range Papillomavirus Transcriptome as a Biomarker of Papillomavirus-Associated Cervical High-Grade Cytology. J Mol Diagn. 2019 Sep;21(5):768-781. Epub 2019 Aug 12). It has been attached here for the sake of the record as it was not in the prior filing.
For the above reasons, the arguments are not considered persuasive.
Conclusion
No claims are allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to EMMA R HOPPE whose telephone number is (703)756-5550. The examiner can normally be reached Mon - Fri 11:00 am - 7:00 pm.
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/EMMA R HOPPE/ Examiner, Art Unit 1683
/ANNE M. GUSSOW/ Supervisory Patent Examiner, Art Unit 1683