Ex Parte Uhlmann et al

Patent Trial and Appeal Board

Jan 17, 2018

10823784 (P.T.A.B. Jan. 17, 2018)

United States Patent and Trademark Office
UNITED STATES DEPARTMENT OF COMMERCE
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APPLICATION NO. FILING DATE FIRST NAMED INVENTOR ATTORNEY DOCKET NO. CONFIRMATION NO.
10/823,784 04/14/2004 Karen Uhlmann 3035-101 4952
46002 7590 01/19/2018
JOYCE VON NATZMER
AGRIS & VON NATZMER LLP
43 West 43rd Street, Suite 104
New York, NY 10036-7424
EXAMINER
HANEY, AMANDA MARIE
ART UNIT PAPER NUMBER
1634
NOTIFICATION DATE DELIVERY MODE
01/19/2018 ELECTRONIC
Please find below and/or attached an Office communication concerning this application or proceeding.
The time period for reply, if any, is set in the attached communication.
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PTOL-90A (Rev. 04/07)
UNITED STATES PATENT AND TRADEMARK OFFICE
BEFORE THE PATENT TRIAL AND APPEAL BOARD
Ex parte KAREN UHLMANN, PETER NURNBERG, and
ANJA BRINCKMANN1
Appeal 2016-006930
Application 10/823,784
Technology Center 1600
Before DEMETRA J. MILLS, ERIC B. GRIMES, and LORA M. GREEN,
Administrative Patent Judges.
GRIMES, Administrative Patent Judge.
DECISION ON APPEAL
This is an appeal under 35U.S.C. § 134 involving claims to a method
for detecting and quantifying methylation of nucleotides, which have been
rejected as obvious. We have jurisdiction under 35 U.S.C. § 6(b).
We affirm.
STATEMENT OF THE CASE
“Methylation of nucleotides such as CpG dinucleotides ... is a key
element of the epigenetic control of genomic information in mammals.”
1 Appellants identify the Real Party in Interest as Max-Delbruck-Centrum
fur Molekulare Medizin. (Br. 5.)
Appeal 2016-006930
Application 10/823,784
(Spec. 1.) “[AJberrant DNA methylation, including hypo- as well as
hypermethylation, is often associated with pathogenesis, such as
tumorigenesis.” (Id.)
“Sodium bisulfite-treatment of DNA converts unmethylated cytosine
into uracil, which is subsequently amplificated [sic] as thymine in a PCR.
Methylated cytosine, however, is non-reactive and remains detectable as a
cytosine.” (Id.) The Specification discloses a method for detecting the
methylation status of predetermined nucleotides. (Id. at 2.) Preferably, “the
method further comprises quantifying the methylated nucleotides.” (Id. at
15.)
Claims 1—5, 7, 9, 11—20, 22, 24, 26—36, 38, 39, and 41—45 are on
appeal. Claim 1 is illustrative and reads as follows:
1. A method for detecting and quantifying the methylation
status of a nucleotide at a predetermined position in a nucleic
acid molecule comprising:
(a) treating a sample comprising said nucleic acid
molecule in water or a buffered solution with an agent suitable
for the conversion of said nucleotide if present in
(i) methylated form; or
(ii) non-methylated form
to pair with a nucleotide normally not pairing with said
nucleotide prior to conversion, wherein non-methylated
cytosine is converted to uracil and methylated cytosine
remains detectable as cytosine;
(b) amplifying said nucleic acid molecule treated with
said agent via at least one amplification primer to
produce an amplification product and converting said
amplification product into single stranded amplified
nucleic acid molecules, wherein said at least one
amplification primer is detectably labeled with a
detectable label that forms an anchor for removal of said
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Application 10/823,784
single stranded amplified nucleic acid molecules to
generate a single stranded amplified nucleic acid
molecule;
(c) real-time sequencing said single stranded amplified
nucleic acid molecule;
(d) detecting whether said nucleotide is methylated or not
methylated at said predetermined position in the sample;
and
(e) quantifying the methylation status by calculating the
frequency of the methylated nucleotides from results of
said real-time sequencing relative to a control nucleic
acid molecule having a known methylation status.
The claims stand rejected as follows:
Claims 1-5, 7-9, 11, 12, 19, 20, 22-25,26-34,36, 39, 41, 43, and 45
under 35 U.S.C. § 103(a) as obvious based on Uhlmann,2 Nyren,3
Markowitz,4 Chen,5 and Eads6 (Final Action7 4);
Claims 12—16, 18, and 38 under 35 U.S.C. § 103(a) as obvious based
on Uhlmann, Nyren, Markowitz, Chen, Eads, and Herman8 (Final Action
12);
2 Karen Uhlmann et al., Changes in methylation patterns identified by two-
dimensional DNA fingerprinting, 20 Electrophoresis 1748—1755 (1999).
3 Nyren, US 6,258,568 Bl, issued July 10, 2001.
4 Markowitz et al., US 2003/0068620 Al, published Apr. 10, 2003.
5 Hong Chen & Barbara Ramsay Shaw, Kinetics of Bisulfite-Induced
Cytosine Deamination in Single-Stranded DNA, 32 Biochemistry 353 5—
3539 (1993).
6 Cindy A. Eads et al., MethyLight: a high-throughput assay to measure
DNA Methylation, 28 Nucleic Acids Research i—viii (1993).
7 Office Action mailed Feb. 20, 2015.
8 Herman et al., US 5,786,146, issued July 28, 1998.
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Claim 17 under 35 U.S.C. § 103(a) as obvious based on Uhlmann,
Nyren, Markowitz, Chen, Eads, Herman, and Feinberg9 (Final Action 13);
Claim 35 under 35 U.S.C. § 103(a) as obvious based on Uhlmann,
Nyren, Markowitz, Chen, Eads, and Faird10 (Final Action 14);
Claim 37 under 35 U.S.C. § 103(a) as obvious based on Uhlmann,
Nyren, Markowitz, Chen, Eads, and Hyman* 11 (Final Action 15); and
Claims 42 and 44 under 35 U.S.C. § 103(a) as obvious based on
Uhlmann, Nyren, Markowitz, Chen, Eads, and Olek12 (Final Action 16).
DISCUSSION
The Examiner has rejected claims 1—5, 7—9, 11, 12, 19, 20, 22—25,
26—34, 36, 39, 41, 43, and 45 as obvious based on Uhlmann, Nyren,
Markowitz, Chen, and Eads. The Examiner finds that Uhlmann discloses a
method meeting most of the limitations of claim 1, but its method does not
include an amplification primer having a label that forms an anchor or real­
time sequencing. (Final Action 4—6.)
The Examiner finds that Nyren discloses a real-time sequencing
method that includes an amplification primer having a label that forms an
anchor for removal of single-stranded nucleic acids. {Id. at 6.) The
Examiner concludes that it would have been obvious to modify Uhlmann’s
method to use Nyren’s sequencing method because Nyren discloses that its
9 Feinberg, US 2003/0232351 Al, published Dec. 18, 2003.
10 Laird et al., US 2002/0086324 Al, published July 4, 2002.
11 Hyman, US 5,602,000, issued Feb. 11, 1997.
12 Olek, US 2005/0019762 Al, published Jan. 27, 2005.
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Application 10/823,784
method allows “DNA to be sequenced simply and rapidly while avoiding the
need for electrophoresis and use of harmful radiolabels.” (Id. at 7.)
The Examiner finds that Uhlmann and Nyren do not teach carrying
out step (a) of claim 1 in water or a buffer solution, but Markowitz and Chen
both teach treating DNA with sodium bisulfite in either water or a buffer
solution. (Id. at 8—9.) The Examiner concludes that it would have been
obvious to carry out Uhlmann’s sodium bisulfite modification step in water
or a buffer solution because that method “was well known, routine, and
conventional in the art.” (Id. at 9.)
Finally, the Examiner finds that Uhlmann, Nyren, Markowitz, and
Chen do not teach quantifying the methylation status of a predetermined
nucleotide, but that “Eads teaches performing bisulfite genomic sequencing
on samples . . . and quantifying the methylation status.” (Id. at 10.) The
Examiner concludes that it would have been obvious
to have modified the method of Uhlmann, Nyren, Markowitz,
and Chen by quantifying the methylation status by calculating
the frequency of the methylated nucleotides from results of
pyrosequencing relative to a control nucleic acid molecule
having a known methylation status as suggested by Eads. One
of skill in the art would have been motivated to quantify the
methylation status of a nucleotide at a predetermined position in
a nucleic acid sample in order to determine how many alleles are
methylated.
(Id. at 11—12.)
We agree with the Examiner that the cited references would have
made obvious the method of claim 1 to a person of ordinary skill in the art.
Uhlmann discloses “determin[ing] the methylation state of distinct
nucleotides within a human DNA fragment of interest using the bisulfite
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approach. This technique is based on sodium bisulfite-mediated conversion
of nonmethylated cytosines to uracil.” (Uhlmann 1749, left col.)
The uracil is amplified as thymine (T) is a PCR reaction. (Id. at 1750,
Figure 1). Uhlmann discloses that after the bisulfite-modified DNA is
amplified by PCR, cloned, and sequenced, “this method reveals the
methylation status of distinct CpGs in individual DNA strands.” (Id.)
Nyren discloses a real-time DNA sequencing method (Nyren 5:61—64)
in which the “sample DNA may be amplified,” (id. at 8:9) and
“[ijmmobilisation of the amplified DNA may take place as part of PCR
amplification itself... or alternatively one or more of the PCR primers may
carry a functional group permitting subsequent immobilisation, eg. a biotin
or thiol group” (id. at 8:21—26). Appellants’ Specification states that the
detectable label recited in claim 1 can be biotin. (Spec. 13.)
We agree with the Examiner that it would have been obvious to a
person of ordinary skill in the art to modify Uhlmann’s method to include
Nyren’s sequencing technique Nyren discloses that its “assay technique is
very simple and rapid, thus making it easy to automate by using a robot
apparatus where a large number of samples may be rapidly analysed.”
(Nyren 8: 61—63.)
Markowitz discloses a method of detecting cancer that includes
reacting DNA with sodium bisulfite to convert non-methylated cytosines to
a different nucleotide. (Markowitz 18.) Markowitz provides a working
example in which DNA was treated with sodium bisulfite after dilution in
distilled water. (Id. 181.) Similarly, Chen describes sodium bisulfite -
induced conversion of cytosine to uracil in a buffer solution containing
deionized water, bisulfite, and Hepes. (Chen 3535, right col.) Thus, it
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Application 10/823,784
would have been obvious to carry out Uhlmann’s process in water or a
buffer solution, rather than in agarose beads, because Markowitz and Chen
disclose that water or a buffer solution is suitable for reacting sodium
bisulfite with DNA to convert non-methylated cytosines to uracil.
Eads discloses “a high-throughput quantitative methylation assay.”
(Eads i, abstract.) Eads discloses that its assay is “highly quantitative and
can very accurately determine the relative prevalence of a particular pattern
of DNA methylation.” (Id.) Eads also discloses that “[t]he gene expression
profile of a cancer specimen is considered a valuable source of biological
information with potential clinical utility” and that “methylation patterns in a
tumor cell are thought to reflect, at least in part, the gene expression profile
of that cell.” (Id. at i, bridging paragraph.) Eads discloses that cytosine
methylation is often associated with transcriptional repression, and
“[tjranscriptional inactivation of CpG island-containing promoters of tumor
suppressor genes by DNA hypermethylation has been well documented in
many human cancers.” (Id.)
Eads discloses that, “[w]hen just the probe is designed to cover CpG
dinucleotides (Fig. 1, application B), then sequence discrimination occurs
solely at the level of probe hybridization” and “the design of separate probes
for each of the different sequence variants associated with a particular
methylation pattern . . . would allow a quantitative determination of the
relative prevalence of each sequence permutation in the mixed pool of PCR
products.” (Id. at iii, right col.) Eads states that application B, which allows
quantitative determination of methylation patterns, “promises to be a
powerful method, since it has the potential to provide quantitative
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information on the relative prevalence of different sequence variants,
representing different methylation patterns.” {Id. at viii, left col.)
We agree with the Examiner that, based on Eads’ teachings, it would
have been obvious to modify the method made obvious by the other cited
references to include a step of quantifying the methylation status of a nucleic
acid sample, because Eads discloses that hypermethylation of tumor
suppressor promoters causes transcriptional inactivation. Thus, those skilled
in the art would reasonably expect that detecting hypermethylation of DNA
from the promoters of tumor suppressor genes would be useful in diagnosing
cancer.
Appellants argue that the Examiner has not identified sufficient reason
to include a quantification step, as required by claim 1, because “[wjanting
to know how much of something (such as alleles) exist... is not more than
a conclusion and not sufficient to show that there was motivation for a
person skilled in the art to combine Uhlmann ‘99, Nyren, Markowitz and
Chen with Eads.” (Br. 17—18.)
We agree with the Examiner, however, that
the skilled artisan would have been motivated to quantify the
methylation status of a nucleotide at a predetermined position in
a nucleic acid sample in order to determine how many alleles are
methylated because the skilled artisan would have recognized
that hyper and/or hypo methylation of alleles (which is what the
quantification is detecting) is indicative of many diseases. The
prior art of Uhlmann and Eads both acknowledge this.
(Ans. 7—8.) As discussed above, Eads discloses that hypermethylation of the
promoters of tumor suppressor genes is associated with transcriptional
repression, and therefore with cancer. (Eads i, bridging paragraph.)
Uhlmann also discloses that cytosine methylation influences gene expression
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and “is recognized as an important factor in tumor development.” (Uhlmann
1749, left col.) Thus, the teachings of the cited references provide a reason
for a person of ordinary skill in the art to include a quantification step in the
method that they collectively made obvious.
Appellants also argue that modifying Uhlmann’s method to carry out
the bisulfite conversion in water or buffer would render it unsatisfactory or
change its principle of operation, because “Uhlmann provides specific
reasons for using agarose beads.” (Br. 15—16.) Appellants argue that
Uhlmann teaches that immobilizing DNA in agarose beads “facilitates the
modification procedure and allows separate PCR reactions of the sense and
antisense strand.” {Id. at 16.)
This argument is unpersuasive, because both Markowitz and Chen
disclose that conversion of unmethylated cytosine to uracil by sodium
bisulfite is carried out effectively in water (Markowitz | 81) or buffer (Chen
3535, right col.). Thus, a skilled artisan would have recognized that
immobilization of DNA in agarose beads is not necessary for bisulfite
modification. In addition, although Uhlmann carries out strand-specific
amplification of DNA after bisulfite treatment (Uhlmann 1750, Fig. 1),
Appellants have not pointed to evidence or provided sound technical
reasoning to show that strand-specific amplification could not be carried out
with DNA in water or buffer (e.g., by dividing an aqueous solution between
two separate tubes).
Appellants also argue that combining Uhlmann’s method with the
pyrosequencing disclosed by Nyren would yield unpredictable results,
because “Nyren only discloses pyrosequencing in the context of not
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Application 10/823,784
chemically treated nucleic acids.” (Br. 16—17.) Appellants cite Nyren’s
Figure 4 and the discussion of that figure in Nyren’s column 19. {Id. at 17.)
We have reviewed the cited figure and discussion in Nyren, but do not
find evidence to support Appellants’ position that Nyren’s method could not
predictably be applied to DNA that had been treated with sodium bisulfite to
convert unmethylated cytosine residues to uracil. The cited evidence simply
does not mention any effect of sodium bisulfite modification on the
effectiveness of Nyren’s pyro sequencing method.
With regard to claims 34 and 39, Appellants argue that Eads states
that its technique was not designed to yield high-resolution methylation
information, and therefore would not have been expected to be capable of
detecting an allele frequency of 5%, as recited in claim 34, or an allele
frequency of 5% with a standard deviation of not more than 1%, as recited in
claim 39. (Br. 18—19.)
As the Examiner pointed out, however, “Eads isn’t being relied upon
for teaching the MethyLight assay. Eads is only being relied upon to teach
the concept of calculating the frequency of methylated nucleotides relative
to control nucleic acids having a known status.” (Ans. 9.) Appellants have
not pointed to evidence or provided sound technical reasoning to support
their position that Uhlmann’s method, as modified by the other cited
references, would not have been capable of detecting a 5% allele frequency,
with or without a standard deviation of 1% or less.
We therefore affirm the rejection of claim 1 under 35 U.S.C. § 103(a)
based on Uhlmann, Nyren, Markowitz, Chen, and Eads. Claims 2—5, 7—9,
11, 12, 19, 20, 22—25, 26—34, 36, 39, 41, 43, and 45 have not been argued
separately and therefore fall with claim 1. 37 C.F.R. § 41.37(c)(l)(iv).
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The Examiner has rejected claims 12—18, 35, 37, 38, 42, and 44 under
35 U.S.C. § 103(a) based on Uhlmann, Nyren, Markowitz, Chen, and Eads,
combined with one or more of Herman, Feinberg, Laird, Hyman, or Olek.
Appellants have waived arguments based on Herman, Feinberg, Laird,
Hyman, or Olek. (See Br. 19.) We therefore affirm the remaining rejections
as well. See 37 C.F.R. § 41.37(c)(l)(iv) (Appeal Brief must contain “[t]he
arguments of appellant with respect to each ground of rejection, and the
basis therefor.”); Hyatt v. Dudas, 551 F.3d 1307, 1314 (Fed. Cir. 2008).
SUMMARY
We affirm all of the rejections on appeal.
TIME PERIOD FOR RESPONSE
No time period for taking any subsequent action in connection with
this appeal may be extended under 37 C.F.R. § 1.136(a).
AFFIRMED
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