IN THE UNITED STATES PATENT AND TRADEMARK OFFICE
`
`____________
`
`BEFORE THE PATENT TRIAL AND APPEAL BOARD
`
`____________
`
`AMBRY GENETICS CORPORATION
`Petitioner
`
`v.
`
`THE JOHNS HOPKINS UNIVERSITY
`Patent Owner
`
`
`
`Case No. To be assigned
`
`U.S. Patent No. 7,824,889
`
`____________________________________
`
`PETITION FOR INTER PARTES REVIEW OF U.S. PATENT NO. 7,824,889
`UNDER 35 U.S.C. §§311-319 AND 37 C.F.R. §§ 42.1-.80, 42.100 ET SEQ.
`
`
`
`
`
`

`

`
`
`INTRODUCTORY STATEMENT ........................................................................... 1
`I. MANDATORY NOTICES (37 C.F.R. § 42.8(a)(1)) ...................................... 1
`A.
`Real Party-in-Interest (37 C.F.R. § 42.8(b)(1)) ..................................... 1
`
`B.
`
`Related Matters (37 C.F.R. § 42.8(b)(2)) .............................................. 1
`
`C. Designation of Lead and Back-Up Counsel (37 C.F.R. §
`42.8(b)(3)) ............................................................................................. 3
`
`A. Notice of Service Information (37 C.F.R. § 42.8(b)(4)) ....................... 3
`
`B. Grounds for Standing (37 C.F.R. § 42.104(a)) ..................................... 3
`
`II.
`
`Payment of Fees (37 C.F.R. § 42.103) .................................................. 4
`C.
`STATEMENT OF THE PRECISE RELIEF REQUESTED AND THE
`REASONS THEREFOR ................................................................................. 4
`III. THE ‘889 PATENT AND ITS TECHNICAL BACKGROUND ................... 5
`IV. A PERSON OF ORDINARY SKILL IN THE ART ...................................... 8
`V.
`CLAIM CONSTRUCTION (37 C.F.R. §§ 42.104(b)(3)) ............................... 9
`A.
`The Preambles Are Not Limiting .......................................................... 9
`
`B.
`
`C.
`
`“to form a set comprising a plurality of assay samples” ..................... 10
`
`“assay samples of the set” ................................................................... 10
`
`D.
`
`“comparing the first number of assay samples to the second
`number of assay samples to ascertain an allelic imbalance in the
`biological sample” ............................................................................... 11
`IDENTIFICATION OF THE CHALLENGE (37 C.F.R. § 42.104(B)) ....... 11
`A. Ground 1: Claim 1 Is Anticipated By Chiang ..................................... 12
`
`VI.
`
`B. Ground 2: Claims 1 and 8 Are Anticipated By Sykes ........................ 17
`
`C. Ground 3: Claims 1 and 8 Would Have Been Obvious In View
`of Chiang and/or Sykes ....................................................................... 24
`VII. CONCLUSION .............................................................................................. 24
`CERTIFICATION TO WORD COUNT UNDER 37 C.F.R. §42.24(d) ................. 26
`
`
`-i-
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`

`

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`
`
`INTRODUCTORY STATEMENT
`
`Ambry Genetics Corporation (“Petitioner” or “Ambry”) in accordance with
`
`35 U.S.C. §§311-319 and 37 C.F.R. §§42.1-.80,41.100-41.123, respectfully
`
`requests inter partes review for claims 1 and 8 of U.S. Patent No. 7,824,889 (‘889
`
`Patent). Petitioner seeks review and cancellation of claims 1 and 8 of the ‘889
`
`Patent. Claims 1 and 8 of the ‘889 Patent are unpatentable as anticipated and/or
`
`obvious under 35 U.S.C. §§ 102 and 103. The U.S. Patent and Trademark Office
`
`(“USPTO”) assignment records indicate that the ‘889 Patent is assigned to The
`
`Johns Hopkins University (“Patent Owner”).
`
`I. MANDATORY NOTICES (37 C.F.R. § 42.8(A)(1))
`
`A. Real Party-in-Interest (37 C.F.R. § 42.8(b)(1))
`
`The real party-in-interest is Ambry Genetics Corporation (“Petitioner” or
`
`“Ambry”). Ambry is a corporation organized under the laws of Delaware.
`
`Petitioner is not barred by operation of estoppel to submit this Petition for inter
`
`partes review.
`
`B. Related Matters (37 C.F.R. § 42.8(b)(2))
`
`U.S. Patent No. 7,824,889 (the ‘889 Patent; Ex. 1001) is asserted against
`
`Petitioner in a pending district court litigation, Esoterix Genetics Laboratories,
`
`LLC and The John Hopkins University v. Ambry Genetics Corporation., United
`
`States District Court for the Middle District of North Carolina, Case No. 1:16-cv-
`
`
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`-1-
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`

`
`
`1111-WO-JEP (the “Ambry litigation”). The complaint was filed on September 7,
`
`2016 (Ex. 1005) and served on Petitioner on September 12, 2016 (Ex. 1006). The
`
`'889 Patent is also presently the subject of a patent infringement lawsuit brought by
`
`the Patent Owner and assignee, The Johns Hopkins University, and its licensee,
`
`Esoterix Genetics Laboratories, against Myriad Genetics, Inc. and Myriad Genetic
`
`Laboratories, Inc., and captioned Esoterix Genetic Laboratories, LLC and The
`
`Johns Hopkins University v. Myriad Genetics, Inc. and Myriad Genetics
`
`Laboratories, Inc., United States District Court for the Middle District of North
`
`Carolina, Case No. 1:16-cv-1112-WE-JEP (the “Myriad litigation”).
`
`Petitioner is concurrently filing petitions for inter partes review of U.S.
`
`Patent Nos. 6,440,706, 7,915,015 and 8,859,206 also owned by Patent Owner and
`
`asserted against Ambry in the Ambry litigation.
`
`Petitioner is aware of a petition for inter partes review of the ‘889 Patent
`
`was filed on 3/16/2017 by Myriad Genetics, Inc., Myriad Genetic Laboratories,
`
`Inc., Bio-Rad Laboratories, Inc., and RainDance Technologies, Inc. This
`
`proceeding was terminated on 8/22/2017.
`
`-2-
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`
`
`C. Designation of Lead and Back-Up Counsel (37 C.F.R. § 42.8(b)(3))
`
`LEAD COUNSEL
`
`BACK-UP COUNSEL
`
`Bhanu K. Sadasivan
`Reg. No. 61,561
`McDermott Will & Emery LLP
`275 Middlefield Rd., Suite 100
`Menlo Park, CA 94025
`
`Jacqueline F. Mahoney
`Reg. No. 48,390
`McDermott Will & Emery LLP
`275 Middlefield Rd., Suite 100
`Menlo Park, CA 94025
`
`A. Notice of Service Information (37 C.F.R. § 42.8(b)(4))
`
`Please address all correspondence to lead and back-up counsel. Petitioner
`
`consents to electronic service by email at: bsadasivan@mwe.com,
`
`Jfmahoney@mwe.com, AmbryEsoterixMWE@mwe.com and
`
`IPdocketMWE@MWE.com.
`
`B. Grounds for Standing (37 C.F.R. § 42.104(a))
`
`Petitioner certifies that the ‘889 Patent is eligible for inter partes review and
`
`further certifies that Petitioner is not barred or otherwise estopped from requesting
`
`inter partes review challenging the identified claims on the grounds in the present
`
`Petition. This Petition is filed within one year of the date Petitioner was served
`
`with a complaint of infringement of the ‘889 Patent. A true copy of the Proof of
`
`Service of Summons and Complaint, showing the date of service of September 12,
`
`2016 is included as Ex. 1006. Petitioner has not filed a civil action challenging the
`
`validity of a claim of the ‘889 Patent. 35 U.S.C. § 315(a).
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`-3-
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`
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`C.
`
`Payment of Fees (37 C.F.R. § 42.103)
`
`The USPTO is hereby authorized to charge Deposit Account No. 505907 for
`
`the fee set in 37 C.F.R. § 42.15(a) for this Petition for inter partes review, and for
`
`any additional fees that may be due as a result of the submission of this Petition.
`
`II.
`
`STATEMENT OF THE PRECISE RELIEF REQUESTED AND THE
`REASONS THEREFOR
`Petitioner respectfully requests inter partes review and cancellation of
`
`claims1 1 and 8 based on 35 U.S.C. §§ 102 and 103 for the reasons stated herein.
`
`This Petition establishes a reasonable likelihood that the Petitioner will prevail in
`
`establishing that the challenged claims are unpatentable. The following chart
`
`summarizes the individual grounds, including the statutory basis and the prior art
`
`relied upon for each ground.
`
`Ground No.
`1
`2
`3
`
`35 USC
`§ 102(b)
`§ 102(b)
`§ 102(b) / § 103
`
`Claims
`1
`1, 8
`1, 8
`
`Prior Art Reference(s)
`Chiang
`Sykes
`Chiang and/or Sykes
`
`“Chiang” (Ex. 1031) was published in 1996 and is prior art to the ‘889
`
`Patent under at least 35 U.S.C. §102(b). “Sykes” (Ex. 1011) was published in
`
`1992 and is prior art to the ‘889 Patent under at least 35 U.S.C. § 102(b).
`
`Chiang was not before the Patent Office during the initial prosecution nor
`
`the ex parte reexamination of the ‘889 Patent. Sykes was nominally before the
`
`
`1 Petitioner does not concede the validity of any of the unchallenged claims.
`-4-
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`

`

`
`
`Patent Office during initial prosecution of the ‘889 Patent. However, Sykes was
`
`not discussed nor used in a prior art rejection during prosecution of the ‘889 Patent.
`
`Ground 1 is not redundant with Ground 2 because Chiang discloses
`
`experiments relating to amplification for different purposes than disclosed in Sykes.
`
`III. THE ‘889 PATENT AND ITS TECHNICAL BACKGROUND
`
`The ‘889 Patent entitled “Digital Amplification” was filed on 2/23/2007 as
`
`Application No. 11/709,742 and issued on 11/2/2010 (Ex. 1001). The ‘889 Patent
`
`claims earliest priority to provisional Application No. 60/146,792 filed 8/2/1999.2
`
`(Id.)
`
`Independent claim 1 recites a method for determining an allelic imbalance in
`
`a biological sample (Ex. 1001). An allelic imbalance is used in the art to refer to
`
`situations in which one allele (of a pair) is expressed at a lower level than the other
`
`due to gene silencing, imprinting, mutations in regulatory sequences, etc., as well
`
`as situations in which one allele is duplicated or deleted from the genome (Buck
`
`Decl., ¶ 34). The method has four basic steps: (1) distributing; (2) amplifying; (3)
`
`analyzing; and (4) comparing. In brief, a sample is partitioned into assay samples,
`
`the template molecules in the assay samples are amplified, the amplified products
`
`
`2 Petitioner is not aware of any claim to an earlier priority date that would affect
`
`any of the arguments set forth herein. Petitioner reserves the right to respond
`
`should Patent Owner assert an earlier priority date.
`
`-5-
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`

`
`
`are analyzed to determine the number of assay samples which contain a “selected
`
`genetic sequence on a first chromosome” or “a reference genetic sequence on a
`
`second chromosome,” the two numbers of assay samples are compared to ascertain
`
`an allelic imbalance in the biological sample.
`
`The ‘889 Patent is entitled “Digital Amplification” and the patentees
`
`distinguish traditional PCR from digital amplification. The patentees summarize
`
`their invention as follows: “The identification of pre-defined mutations expected
`
`to be present in a minor fraction of a cell population is important for a variety of
`
`basic research and clinical applications. The exponential, analog nature of the
`
`polymerase chain reaction is transformed into a linear, digital signal suitable for
`
`this purpose. Single molecules can be isolated by dilution and individually
`
`amplified; each product is then separately analyzed for the presence of pre-defined
`
`mutations. This process provides a reliable and quantitative measure of the
`
`proportion of variant sequences within a DNA sample.” (‘889 Patent, Abstract.)
`
`The claimed methods, however, are not restricted to the process the
`
`patentees summarize as their invention. Moreover, the Patent Owner has proffered
`
`a much broader definition of the scope of the claims in the litigation over the ‘889
`
`Patent at the district court. In essence, the Patent Owner is contending that a
`
`traditional PCR reaction conducted in duplicate followed by sequencing or another
`
`detection method would meet the claimed methods. Specifically, the claimed
`
`methods, according to the Patent Owner:
`-6-
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`

`
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`(1) are not limited to single molecules isolated by dilution;
`
`(2) the number of assay samples can be just two;
`
`(3) the molecules that are amplified are not limited to the “selected genetic
`
`sequence” or “reference genetic sequence”;
`
`(4) the assay samples with the amplified molecules need not even be part of
`
`the claimed set- they may simply be “associated” with the set;
`
`(5) the “first number of assay samples” which contain the “selected genetic
`
`sequence” and the “second number of assay samples” with contain the “reference
`
`genetic sequence” can be just one each;
`
`(6) the first number need not be “compared to” the second number,
`
`“examining” the number such as by sequencing the amplified product is sufficient;
`
`and
`
`(7) “an allelic
`
`imbalance
`
`in
`
`the biological sample” need not be
`
`“ascertained.” Simply attempting to “discover” an allelic imbalance (even if one
`
`had no intention of ascertaining an allelic imbalance) would meet this limitation.
`
`Thus, under the claim construction proffered by the Patent Owner the
`
`claimed methods would be taught by traditional PCR followed by well-known
`
`detection methods to identify allelic imbalance, such as that taught by Chiang (Ex.
`
`1031).
`
`In any event, “digital amplification” as disclosed in the ‘889 Patent was well
`
`known in the art for about a decade prior to the earliest priority date for the ‘889
`-7-
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`

`
`
`Patent. (Exs. 1012-1013, 1015-1025, Buck Decl., ¶¶ 28-29.) In the prior art, this
`
`method was often called “single molecule PCR,” “limiting dilution analysis” or
`
`“limiting dilution PCR” (“LDPCR”). Sykes and others in 1992 had disclosed the
`
`concept of digital PCR (Ex. 1011 and 1017). The steps of diluting and distributing
`
`template for use in quantitative PCR were so well-known in the art that it was
`
`published as a book chapter (Ex. 1016). Indeed, multiple groups were performing
`
`the steps and publishing their results before the filing of the asserted patent
`
`applications (Ex. 1010, 1011, 1021-1024).
`
`In 1990, Simmonds described the use of LDPCR to quantify HIV as the
`
`target of interest (Ex. 1009). LDPCR was further used to study various aspects of
`
`treatment and biology of acute lymphoblastic leukemia (ALL) prior to the priority
`
`date of the ‘889 Patent (Ex. 1011). Patent Owner did nothing more than what was
`
`known in the prior art method of LDPCR.
`
`IV. A PERSON OF ORDINARY SKILL IN THE ART
`
`With respect to the ‘889 Patent, a person of ordinary skill in the art
`
`(“POSA”) is a hypothetical person who is presumed to be aware of all pertinent art,
`
`thinks along the lines of the conventional wisdom in the art, and is a person of
`
`ordinary creativity.
`
`As of August 2, 1999, a POSA would typically have earned a Master’s
`
`degree in the biological sciences or a related field, and have at least four years of
`
`molecular biology laboratory experience, or alternatively, have a Ph.D. degree in
`-8-
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`
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`the biological sciences or a related field, and have at least two years of molecular
`
`biology laboratory experience. (Buck Decl., ¶ 12.)
`
`V. CLAIM CONSTRUCTION (37 C.F.R. §§ 42.104(B)(3))
`
`In accordance with 37 C.F.R. § 42.100(b), the challenged claims must be
`
`given their broadest reasonable interpretations (BRI) in light of the specification
`
`and prosecution history of the ‘889 Patent. Since the ‘889 Patent Reexamination
`
`Certificate was issued, Patent Owner has asserted the challenged ‘889 Patent
`
`claims against certain of Ambry's tests and services that are multi-gene panels that
`
`identify an elevated risk for multiple types of cancers. (Ex. 1005.) In that
`
`litigation, Plaintiff has proposed a broad construction for the challenged claims, as
`
`explained below. Petitioner submits that the claims are invalid under the broad
`
`construction of claims proposed by Patent Owner.
`
`A. The Preambles Are Not Limiting
`
`Under the BRI, the preamble of claim 1 should not be limiting. This
`
`preamble does not recite any structure or step needed to give meaning and life to
`
`the claims, or to any dependent claims. See, e.g., Summit 6, LLC v. Samsung
`
`Electronics Co., Ltd., 802 F.3d 1283, 1292 (Fed. Cir. 2015) (“[g]enerally, a
`
`preamble is not limiting”); TomTom, Inc. v. Adolph, 790 F.3d 1315, 1323 (Fed.
`
`Cir. 2015). No term in claim 1 or in claim 8 that depends from it, refers back to
`
`this preamble, which therefore do not provide any antecedent basis for the body of
`
`the claims. A POSA would have understood that this preamble merely recites an
`-9-
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`

`
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`intended use of the claimed methods, and therefore does not limit the claims in any
`
`way. Summit, 802 F.3d at 1292; TomTom, 790 F.3d at 1323 (Ex. 1015, 1016). To
`
`the extent that the preamble is limiting, it is disclosed by the prior art as set forth
`
`below.
`
`B.
`
`“to form a set comprising a plurality of assay samples”
`
`The method of independent claim 1 requires that the nucleic acid template
`
`molecules be diluted/ distributed from a biological sample “to form a set
`
`comprising a plurality of assay samples.” (Ex. 1001.) Patent Owner’s proposed
`
`construction of the language is “to form a group of two or more portions of the set
`
`for analysis, each of which may or may not comprise one or more nucleic acid
`
`template molecules.” (Ex. 1014.) Under Patent Owner’s definition, the assay
`
`samples in a set may be just two, i.e., duplicates that are analyzed.
`
`C.
`
`“assay samples of the set”
`
`Patent Owner proposed “amplified molecules in the assay samples of the
`
`set” (in the amplification step) to mean “amplified molecules included, located, or
`
`positioned within the assay samples associated with the set.” (Ex. 1014.) Under
`
`this proposed construction, the amplified molecules need not even be part of the
`
`set, but merely “associated with” the set.
`
`-10-
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`

`
`
`D.
`
`“comparing the first number of assay samples to the second
`
`number of assay samples to ascertain an allelic imbalance in the
`
`biological sample”
`
`Patent Owner proposed the language “comparing the first number of assay
`
`samples to the second number of assay samples to ascertain an allelic imbalance in
`
`the biological sample” should be construed as “examining the first number and the
`
`second number in order to discover an allelic imbalance in the biological sample
`
`(the biological sample from which the template molecules were isolated” (Ex.
`
`1014). A sample can be “separated” or “spread out” without requiring dilution.
`
`Under Patent Owner’s proposed construction, simply examining two sequences
`
`(such as after sequencing) without any comparison would meet this claim
`
`limitation. (Buck Decl., ¶ 43.)
`
`VI.
`
`IDENTIFICATION OF THE CHALLENGE (37 C.F.R. § 42.104(B))
`
`Petitioner respectfully petitions for inter partes review of claims 1 and 8 of
`
`the ‘889 Patent based on the unpatentability grounds summarized in the index
`
`below. Per 37 C.F.R. § 42.6(c), copies of the cited references accompany this
`
`Petition.
`
`-11-
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`

`
`
`35 USC
`Claims
`Prior Art Reference(s)
`Ground No.
`1
`§ 102(b)
`1
`Chiang
`2
`§ 102(b)
`1, 8
`Sykes
`3
`§ 102(b) / § 103
`1, 8
`Chiang and/or Sykes
`A. Ground 1: Claim 1 is Anticipated By Chiang
`
`As illustrated in the claim charts and discussion below, a POSA would have
`
`understood that Chiang discloses each element of, and therefore anticipates, Claim
`
`1 of the ‘889 Patent.
`
`Chiang reports using fluorescent-PCR reaction to detect genomic sequence
`
`copy number and transcriptional abundance. (Ex. 1031, 1013 (abstract).) An
`
`alteration in genomic sequence copy number can be used to determine allelic
`
`imbalance. (Buck Decl., ¶ 39.)
`
`Chiang discloses the strategy of their assay as follows: Two PCR reactions
`
`were performed in parallel on both the unknown and the reference samples, i.e. at
`
`least four assay samples. One of the PCR reaction employs primers from the
`
`experimental S100β marker on chromosome 21 (i.e., the “selected genetic
`
`sequence on a first chromosome”) and the other PCR reaction employs primers
`
`from the reference IGF-1 marker on chromosome 12 (i.e., the “reference genetic
`
`sequence on a second chromosome”). (1014.) The PCR reactions with the
`
`different primers were monitored and compared. (Id.) “Because both reactions
`
`were monitored in exponential phase, the PCR signal depended directly on the
`
`initial target concentration. Under these conditions, the ratio of PCR signal from
`
`-12-
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`

`

`
`
`the two reactions (S100β /IGF-1) in the unknown DNA, divided by the ratio of
`
`PCR signal from the two reactions (S100β /IGF-1) in the control (normal
`
`placental) DNA, accurately reflected the relative dosage of S100β to IGF-1 in the
`
`unknown DNA.” (Id.) (Buck Decl., ¶ 40.)
`
`Chiang further discloses using this strategy to determine allelic imbalance in
`
`five samples, Del21ILS, Dup21NA, Del6918, Dup21WB and Del21DS “with
`
`segmental aneusomy for chromosome 21.” (1015.) Three different twofold
`
`dilutions were made of DNA from each of the five samples and a normal control
`
`(placenta) and amplified (1014), thereby meeting steps (a) and (b). (Buck Decl., ¶
`
`41.)
`
`Chiang teaches that the PCR reactions with experimental S100β marker on
`
`chromosome 21 (i.e., “a first number of assay samples which contain a selected
`
`genetic sequence on a first chromosome”) and the PCR reactions with the
`
`reference IGF-1 marker on chromosome 12 (i.e., “a second number of assay
`
`samples which contain a reference genetic sequence on a second chromosome”)
`
`were monitored and the PCR signals measured, thereby meeting step (c). Further,
`
`because three different twofold dilutions were made of DNA from each of the five
`
`samples and the control (1014), and “[q]uantitative analysis requires
`
`reproducibility, so that each reaction was repeated multiple times” (1023), Chiang
`
`also discloses the limitation “between 0.1 and 0.9 of the assay samples yield an
`
`-13-
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`

`
`
`amplification product of at least one of the selected and the reference genetic
`
`sequences.” (Buck Decl., ¶ 42.).
`
`Chiang discloses ascertaining allelic imbalance in a biological sample and
`
`thereby meets step (d). (See 1015 (“The copy numbers of the S100β marker on
`
`chromosome 21 derived in a blinded fashion were three for samples Dup21WB
`
`and Dup21DS, two for samples Dup21NA and Del6918 and one for sample
`
`Del21ILS (Table 1).”); Buck Decl., ¶ 44.)
`
`The claim chart below provides the disclosures in Chiang that disclose each
`
`element of claim 1 of the ‘889 patent:
`
`Claim
`1. A method for
`determining an allelic
`imbalance in a biological
`sample, comprising the
`steps of:
`
`Disclosure in Chiang
`“Table 1. Quantitative Analysis of Samples with
`Aneusomy for Chromosome 21 Using the
`AmpliSensor Method” (1014)
`
`“Two PCR reactions were performed in parallel on
`both the unknown and the reference samples. In
`Table 1, primers from the experimental S100β
`marker on chromosome 21 were used for one PCR
`reaction and compared with the PCR reaction
`achieved with primers from the reference (eusomic)
`IGF-1 marker on chromosome 12. Because both
`reactions were monitored in exponential phase, the
`PCR signal depended directly on the initial target
`concentration. Under these conditions, the ratio of
`PCR signal from the two reactions (S100β /IGF-1) in
`the unknown DNA, divided by the ratio of PCR
`signal from the two reactions (S100β /IGF-1) in the
`control (normal placental) DNA, accurately reflected
`the relative dosage of S100β to IGF-1 in the
`unknown DNA.” (1014)
`
`
`-14-
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`

`

`
`
`Claim
`
`(a) distributing isolated
`nucleic acid template
`molecules to form a set
`comprising a plurality of
`assay samples, wherein the
`nucleic acid template
`molecules are isolated from
`the biological sample;
`
`(b) amplifying the template
`molecules within the set to
`form a population of
`amplified molecules in
`individual assay samples of
`the set;
`(c) analyzing the amplified
`molecules in the assay
`samples of the set to
`determine a first number of
`assay samples which
`contain a selected genetic
`sequence on a first
`chromosome and a second
`number of assay samples
`
`Disclosure in Chiang
`“This enabled us to calculate that sample De121ILS
`had one copy of S100β, samples Dup21NA and
`Del6918 had two copies of S100β; and samples
`Dup21WB and Dup21DS had three copies of
`S100β.” (1014)
`
` “Quantitative analysis requires reproducibility so
`that each reaction was repeated multiple times.”
`(1023)
`
`“PCR was performed in a 96-well plate (PTC-100,
`MJ Research)” (1023)
`
`“In each case, at least two different measurements at
`each of three different twofold dilutions were taken
`in both normal control (placental) DNA and DNA
`from the cells under investigation.” (1014)
`
`“The genomic DNAs were in the range of 5 X 102 to
`1 X 104 copies per μl of the DNA sequence to be
`amplified, using 50 ng/μl of yeast tRNA as the
`dilution solution. An aliquot of 2.5 μL from each
`dilution was used for PCR.” (1023)
`
`“Two PCR reactions were performed in parallel on
`both the unknown and the reference samples.”
`(1014)
`
`“PCR was performed in a 96-well plate (PTC-100,
`MJ Research)” (1023)
`“In Table 1, primers from the experimental S100β
`marker on chromosome 21 were used for one PCR
`reaction and compared with the PCR reaction
`achieved with primers from the reference (eusomic)
`IGF-1 marker on chromosome 12. Because both
`reactions were monitored in exponential phase, the
`PCR signal depended directly on the initial target
`concentration. Under these conditions, the ratio of
`PCR signal from the two reactions (S100β /IGF-1) in
`
`-15-
`
`

`

`
`
`Claim
`which contain a reference
`genetic sequence on a
`second chromosome,
`wherein between 0.1 and
`0.9 of the assay samples
`yield an amplification
`product of at least one of
`the selected and the
`reference genetic
`sequences;
`
`Disclosure in Chiang
`the unknown DNA, divided by the ratio of PCR
`signal from the two reactions (S100β /IGF-1) in the
`control (normal placental) DNA, accurately reflected
`the relative dosage of S100β to IGF-1 in the
`unknown DNA.” (1014)
`
`“This enabled us to calculate that sample De121ILS
`had one copy of S100β, samples Dup21NA and
`Del6918 had two copies of S100β; and samples
`Dup21WB and Dup21DS had three copies of
`S100β.” (1014)
`
`
`
`“The genomic DNAs were in the range of 5 X 102 to
`1 X 104 copies per μl of the DNA sequence to be
`amplified, using 50 ng/μl of yeast tRNA as the
`dilution solution. An aliquot of 2.5 μL from each
`dilution was used for PCR.” (1023)
`
`“Quantitative analysis requires reproducibility so
`that each reaction was repeated multiple times.”
`(1023)
`
`See Claim 1, step (c)
`
`-16-
`
`(d) comparing the first
`number of assay samples to
`
`

`

`
`
`Claim
`the second number of assay
`samples to ascertain an
`allelic imbalance in the
`biological sample
`
`
`Disclosure in Chiang
`
`B. Ground 2: Claims 1 and 8 Are Anticipated By Sykes
`
`As illustrated in the claim charts and discussion below, a POSA would have
`
`understood that Sykes discloses each element of, and therefore anticipates, Claims
`
`1 and 8 of the ‘889 Patent.
`
`1.
`
`Independent Claim 1
`
`Sykes describes “a general method to quantitate the total number of initial
`
`targets present in a sample using limiting dilution, PCR and Poisson statistics.”
`
`(Ex. 1011, abstract). “[A]pproximately two potentially amplifiable leukemic IgH
`
`targets could be detected in the presence of 160,000 competing non-leukemic
`
`genomes” by using the method disclosed in Sykes (Ex. 1011; Abstract). (Buck
`
`Decl., ¶ 47.)
`
`To the extent the preamble is limiting, Sykes discloses a method for
`
`determining an allelic imbalance in a biological sample. (Buck Decl., ¶ 48.)
`
`Specifically, Sykes discloses the “…detection of rare leukemic cells in a large
`
`population of normal cells, which in molecular terms became the problem of
`
`detection of a rare unique IgH sequence against a background of numerous other
`
`IgH sequences.” (Ex. 1011; 448.) For a normal sample, the ratio of any specific
`
`-17-
`
`

`

`
`
`leukemic rearranged IgH sequence to N-ras is expected to be extremely low. A
`
`leukemic sample, on the other hand, will consist predominantly of one specific
`
`leukemic rearranged IgH sequence, e.g., the Ho DNA disclosed in Sykes (Ex.
`
`1011; 445). The presence of a single, dominant, rearranged IgH sequence
`
`represents an allelic imbalance of the IgH gene region. (Buck Decl., ¶¶ 48-50.)
`
`The rearranged immunoglobulin heavy chain (IgH) gene from a leukemic
`
`clone Ho was used as the target DNA. (444-445). Serial dilutions of 10 replicates
`
`of Ho DNA were made and amplified by PCR. (446). 10 replicates were tested for
`
`IgH and 10 replicates were tested for N-ras. (Table 1, 446). “PCR amplification
`
`of N-ras gene was used as an internal control to quantitate the number of
`
`potentially amplifiable genomes present in a sample.” (Abstract). Thus, Sykes
`
`discloses diluting Ho DNA to form a set of at least 20 assay samples and thereby
`
`meets step (a). (Buck Decl., ¶ 51.)
`
`The IgH gene and N-ras gene in the Ho DNA in the assay samples were
`
`amplified by PCR and thereby meets step (b). (Buck Decl., ¶ 52).
`
`The amplified IgH and N-ras products in the assay samples of the set were
`
`analyzed by counting the number of assay samples with IgH and the number of
`
`assay samples with N-ras and thus meets step (c). For example, at a certain
`
`dilution, 6/10 tubes scored positive for N-ras, while 3/10 tubes scored positive for
`
`IgH (Table 1). Sykes also discloses using Poisson statistics to quantitate the initial
`
`numbers of leukemic and non-leukemic templates present. (Abstract, 444, 446-
`-18-
`
`

`

`
`
`447; Buck Decl., ¶ 53.)
`
`The number of assay samples containing the leukemic templates (IgH) were
`
`compared with the number of assay samples containing the total templates, to
`
`ascertain a ratio (“Ratio N-ras/IgH,” Table 2) which reflects an allelic imbalance of
`
`the biological sample (i.e., Ho DNA). The Ho DNA disclosed in Sykes consists of
`
`a single, dominant rearranged IgH sequences and represents an allelic imbalance of
`
`the IgH gene region. Sykes thereby discloses step (d). (Buck Decl., ¶¶ 54-55.)
`
`Claim
`1. A method for determining
`an allelic imbalance in a
`biological sample,
`comprising the steps of:
`
`Disclosure in Sykes
`This non-limiting preamble is nonetheless
`disclosed:
`“We describe a general method to quantitate the
`total number of initial targets present in a sample
`using limiting dilution, PCR and Poisson statistics.
`The DNA target for the PCR was the rearranged
`immunoglobulin heavy chain (IgH) gene derived
`from a leukemic clone that was quantitated against
`a background of excess rearranged IgH genes from
`normal lymphocytes. The PCR was optimized to
`provide an all-or-none endpoint at very low DNA
`target numbers. PCR amplification of the N-ras
`gene was used as an internal control to quantitate
`the number of potentially amplifiable genomes
`present in a sample and hence to measure the extent
`of DNA degradation. A two-stage PCR was
`necessary owing to competition between leukemic
`and non-leukemic templates. Study of eight
`leukemic samples showed that approximately
`two potentially amplifiable leukemic IgH targets
`could be detected in the presence of 160,000
`competing non-leukemic genomes.” (Abstract)
`
`“The biological problem in our study was the
`detection of rare leukemic cells in a large
`
`-19-
`
`

`

`
`
`Claim
`
`(a) distributing isolated
`nucleic acid template
`molecules to form a set
`comprising a plurality of
`assay samples, wherein the
`nucleic acid template
`molecules are isolated from
`the biological sample;
`
`Disclosure in Sykes
`population of normal cells, which in molecular
`terms became the problem of detection of a rare
`unique IgH sequence against a background of
`numerous other IgH sequences. The two-stage PCR
`system that was developed proved capable of
`detecting approximately two (1/0.52) potentially
`amplifiable leukemic IgH sequences against a
`background of approximately 160,000 total
`genomes. These genomes would provide a vast
`excess of sequences that would compete with the
`leukemic IgH sequences for the PCR primers
`because they would contain approximately 2.4x104
`
`rearranged IgH genes from normal B lymphocytes
`and 3x105
` germ-line IgH genes, each containing
`multiple V and J segments.” (448)
`
`“Nevertheless, as seen in Table 2, in all 8 patients
`there was an approximately constant ratio between
`the number of amplifiable IgH targets and the
`number of amplifiable N-ras targets. These data
`suggest that the number of amplifiable N-ras genes,
`rather than the DNA concentration, is the best
`indicator of the number of amplifiable genomes
`present, that virtually all potentially amplifiable
`leukemic IgH genes are amplified in the absence of
`competing non-leukemic IgH genes and that
`approximately half of the leukemic IgH genes are
`amplified in the presence of competing genes.”
`(448)
`“. . . we have used the principle of limiting dilution,
`which is based on the use of a qualitative all-or-
`none end point and on the premise that one or more
`targets in the reaction mixture give rise to a positive
`end point.