Ex Parte Mohammed

Board of Patent Appeals and Interferences

Sep 28, 2007

10260733 (B.P.A.I. Sep. 28, 2007)

The opinion in support of the decision being entered
today is not binding precedent of the Board.

UNITED STATES PATENT AND TRADEMARK OFFICE
____________________

BEFORE THE BOARD OF PATENT APPEALS
AND INTERFERENCES
____________________

Ex parte MANSOOR MOHAMMED
____________________

Appeal 2007-3395
Application 10/260,7331
Technology Center 1600
____________________

Decided: 28 September 2007
____________________

Before RICHARD E. SCHAFER, TEDDY S. GRON and
CAROL A. SPIEGEL, Administrative Patent Judges.

SPIEGEL, Administrative Patent Judge.

DECISION ON APPEAL
I. Introduction
Mansoor Mohammed (hereinafter "Appellant") seeks our review
under 35 U.S.C. § 134 (2002) of the Examiner's final rejection of all pending
claims in this Application, claims 1-3, 5-7, 13-14, 18-25, 28-31, 34-41, 46-

1 Application filed 27 September 2002. Applicant claims benefit under 35
U.S.C. § 119 of application 60/325,853, filed 27 September 2001. The real
party-in-interest is said to be PerkinElmer LAS, Inc.
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49, 54-58, 61-62 and 86. We have jurisdiction under 35 U.S.C. § 6(b)
(2002). We REVERSE.
The subject matter on appeal is directed to a method of detecting the
presence of two or more chromosomally distinct cell lines within a
population of cells using array-based comparative genomic hybridization
("CGH"). Claim 12 is illustrative and reads as follows (emphasis added):
1. A method of detecting a degree of genetic
mosaicism in a cell population by performing an
array-based comparative genomic hybridization
(CGH), wherein an array comprising a plurality of
cloned genomic nucleic acid segments is provided
in a plurality of identical replicas, each cloned
segment immobilized to a discrete and known spot
on a substrate surface to form the array, the cloned
genomic nucleic acid segments comprising a
substantially complete first genome of a known
first karyotype, the method comprising:
(a) contacting replicas of the array with
mixtures of a first nucleic acid sample and a
second nucleic acid sample and fractional dilutions
of the second sample, wherein the first sample
comprises a plurality of genomic nucleic acid
segments comprising a substantially complete
complement of the first genome labeled with a first
detectable label, the second sample comprises a
plurality of genomic nucleic acid segments
comprising a substantially complete complement
of the second genome labeled with a second
detectable label, and the karyotype of the second
sample is known and is different from that of the
first sample;

2 Amended Appeal Brief under 37 C.F.R. § 41.37 filed 27 November 2006
("Br."), at 10.
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(b) contacting further replicas of the array
with mixtures of the first nucleic acid sample and a
third nucleic acid sample and fractional dilutions
of the third sample, wherein the third sample
comprises a genomic nucleic acid sample with an
unknown karyotype and is labeled with the second
detectable label, and the genomic nucleic acid of
the third sample comprises a substantially
complete complement of genomic nucleic acid of a
third genome from a test cell or a tissue sample,
wherein the contacting is under conditions wherein
the nucleic acid in the mixtures of each of the first
and second samples and the first and third samples
can specifically hybridize to the genomic nucleic
acid segments immobilized on the array;
(c) measuring the amount of first label and
second label on each spot for each respective
contacted array and determining the karyotype of
each dilution fraction by comparative genomic
hybridization; and,
(d) selecting which fractional dilution
karyotype determination of the second sample
most closely determines the known karyotype, and
selecting data for the same fractional dilution of
the third sample to determine the karyotype of the
third sample, thereby determining the degree of
genetic mosaicism in the cell population.
Claim 34 limits the array-immobilized genome of the method of claim 1 to a
wild-type (normal) genome, while claim 35 limits the first sample of the
method of claim 34 to a wild-type (normal) genome. Claim 37 requires the
second sample of the method of claim 1 to have a mosaic karyotype. [Br. at
13.]
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The Examiner has rejected claims 1-3, 5-7, 13-14, 18-25, 28-31, 34-
41, 46-49, 54-58, 61-62 and 86 under 35 U.S.C. § 103(a) (Answer3 at 3 and
7). The Examiner relies upon the following prior art4 of record:
Bradley US 6,048,695 Apr. 11, 2000
Boa US 6,251,601 B1 Jun. 26, 2001

Kuukasjärvi, "Optimizing DOP-PCR for Universal Amplification of
Small DNA Samples in Comparative Genomic Hybridization," Genes,
Chromosomes & Cancer, Vol. 18, pp. 94-101 (1997).

Bradley, Bao and Kuukasjärvi qualify as prior art under 35 U.S.C. § 102(b).
According to the Examiner, claims 1-3, 5-7, 13-14, 18-25, 28-31, 34-41, 46,
54-58, 61-62 and 86 would have been obvious over the combined teachings
of Bao and Kuukasjärvi; and, claims 47-49 would have been obvious over
the combined teaches of Bao, Kuukasjärvi, and Bradley.
According to Appellant, the patentability of all the claims on appeal
stand or fall with claim 1 (Br. at 5 and 8). Therefore, we decide this appeal
on the basis of claim 1. 37 C.F.R. § 41.37(c)(1)(v).
II. Findings of Fact ("FF")
The following findings of fact are supported by a preponderance of
the evidence of record.
A. Appellant's specification
[1] CGH is said to be a molecular cytogenetics approach that can be used
to detect regions in a genome undergoing quantitative changes, e.g.,
gains or losses of sequence or copy numbers (Specification at 28, ¶
85).
[2] According to the specification, the

3 Examiner's Answer mailed 12 March 2007 ("Answer").
4 No references to et al. are made in this opinion.
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principal of the array CGH approach is simple.
Equitable amounts of total genomic DNA from
cells of a test sample and a reference sample (e.g.,
a sample from cells known to be free of
chromosomal aberrations) are differentially labeled
with fluorescent dyes and co-hybridized to the
array of BACs [bacterial artificial chromosomes],
which contain the cloned genomic DNA fragments
that collectively cover the cell's genome. The
resulting co-hybridization produces a fluorescently
labeled array, the coloration of which reflects the
competitive hybridization of sequences in the test
and reference genomic DNAs to the homologous
sequences within the arrayed BACs.
Theoretically, the copy number ratio of
homologous sequences in the test and reference
genomic DNA samples should be directly
proportional to the ratio of their respective
fluorescent signal intensities at discrete BACs
within the array. [Specification at 2, ¶ 4, bracketed
text added.]
[3] Further according to the specification, "[t]he methods of the invention
are used to determine the karyotype of a cell population, which
includes an [sic] determination of the genetic mosaicism of a cell
population, including the number of karyotype subpopulations in a
sample and the percent of the cell population having a particular
karyotype" (Specification at 18, ¶ 55).
[4] Genetic mosaicism is said to be defined as "the presence of two or
more chromosomally distinct cell lines or cell lineages within a
sample or a reference population of cells. For example, a solid
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tumor's ('a sample') genetic make-up can be 50% 47,XXX and 50% 45
X,-X cells." (Specification at 2, ¶ 5).5
[5] Determining the presence or degree of genetic mosaicism in a cell
population can be helpful in determining the cause of a disease (e.g.,
cancer or an inherited disease) or for diagnosing or prognosing its
cause (Specification at 2, ¶ 5).
[6] Instead of analyzing the chromosomes from individual cells,
karyotyping using array-based CGH analyzes the DNA sequence copy
number of the total genomic DNA extracted from the cells
(Specification at 2, ¶ 6).
[7] From a DNA copy number perspective, the genome profile of a tumor
made up of 50% 47,XXX cells and 50% 45 X,-X cells is said to be no
different from a reference population of 100% 46,XX cells
(Specification at 2-3, ¶ 6).
[8] However, "the genetic mosaicisms observed in clinical samples will
likely only rarely involve cell populations whose combined genetic
profiles completely mask the presence of a mosaic population"
(Specification at 3, ¶ 7).
[9] The methods described in Appellant's specification are said to be
"sufficiently sensitive to detect clonally distinct (by karyotypic
criteria) cell populations within a more dominant background cell
population" (Specification at 14, ¶ 42).

5 The standard way of describing karyotypic information is to (a) give the
total number of chromosomes, (b) identify the sex chromosomes and (c)
identify any other abnormalities that may be present. A normal male
karyotype, 46,XY, means the cell contains 46 chromosomes, including one
X and one Y.
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[10] The methods described in Appellant's specification are said to
determine "the number of karyotype subpopulations in a sample and
the percent of the cell population having a particular karyotype"
(Specification at 18, ¶ 55).
[11] In one embodiment, the specification describes a method comprising
(a) providing an array comprising a plurality of
cloned genomic nucleic acid segments, wherein
each genomic nucleic acid segment is immobilized
to a discrete and known spot on a substrate surface
to form an array and the cloned genomic nucleic
acid segments comprise a substantially complete
first genome of a known karyotype; (b) providing
a first sample, wherein the sample comprises a
plurality of genomic nucleic acid segments
comprising a substantially complete complement
of the first genome labeled with a first detectable
label; (c) providing a second sample, wherein the
sample comprises a plurality of genomic nucleic
acid labeled with a second detectable label, and the
genomic nucleic acid sample comprises a
substantially complete complement of genomic
nucleic acid of a cell or a tissue sample, and the
karyotype of the second sample is known and is
different from that of the first sample of step (b);
(d) providing a third sample, wherein the sample
comprises a genomic nucleic acid sample with an
unknown karyotype labeled with the second
detectable label, and the genomic nucleic acid
comprises a substantially complete complement of
genomic nucleic acid of a cell or a tissue sample;
(e) preparing serial dilution fractions of the
samples of steps (c) and (d); (f) contacting the
sample of step (b) separately with each serial
dilution fraction of the sample of step (c) with the
array of step (a) under conditions wherein the
nucleic acid in the samples can specifically
hybridize to the genomic nucleic acid segments
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immobilized on the array; (g) measuring the
amount of first and second fluorescent label on
each spot after the contacting of step (f) for each
serial dilution fraction and determining the
karyotype of each serial dilution fraction by
comparative genomic hybridization; (h)
contacting the sample of step (b) and serial dilution
fractions of the sample of step (d) with the array of
step (a) under conditions wherein the nucleic acid
in the samples can specifically hybridize to the
genomic nucleic acid segments on the array; (i)
measuring the amount of first and second
fluorescent label on each spot after the contacting
of step (h) for each serial dilution fraction and
determining the karyotype of each serial dilution
fraction by comparative genomic hybridization;
and, (j) selecting which dilution fraction
karyotype determination of step (g) most closely
determined the known karyotype, and selecting the
same serial dilution measurement in step (i) to
determine the karyotype of the sample of step (d),
thereby determining the degree of genetic
mosaicism in a cell population. [Specification at
4-5, ¶ 12.]
[12] The array-immobilized genome may comprise a normal genome or
karyotype, and the first sample may have a normal genome or
karyotype (Specification at 6-7, ¶ 21).
[13] The second sample may have a mosaic karyotype comprising two or
more cell subpopulations (Specification at 7, ¶ 22).
B. Bao
[14] Chromosomal abnormalities, which may or may not involve a change
in DNA sequence copy number from the normal one copy per
chromosome, are said to be involved in various human pathologies
(Bao at col. 1, ll. 24-32).
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[15] One form of aggressive breast cancer is said to result from gene
amplification and overexpression of the Her-2/neu gene located on
chromosome 17 at band q12 (Bao at col. 1, ll. 35-38).
[16] Overexpression of the Her-2 gene, prior to gene amplification, is said
to occur at an earlier, less aggressive stage of the disease (Bao at col.
1, ll. 42-46).
[17] Thus, proper management of breast cancer is said to require both
measurement of Her-2 gene expression and Her-2 gene copy number,
i.e., two separate tests on a tissue sample (Bao at col. 1, ll. 46-49).
[18] Bao describes methods for simultaneously measuring gene expression
and chromosome abnormalities in the same tissue sample using array-
based CGH (Bao at col. 2, l. 57 to col. 3, l. 3; col. 6, ll. 23-28).
[19] The Bao method comprises cohybridizing first, second and at least
one reference nucleic acid populations, each labeled with a different
fluorescent marker, to an array of nucleic acid target elements
immobilized on a solid substrate and measuring the presence and
intensity of each marker at each target element of the array (Bao at
col. 2, l. 66 through col. 3, l. 12; col. 3, ll. 31-55).
[20] The nucleic acid target elements may comprise total genomic DNA6
(Bao at col. 7, l. 33 through col. 8, l. 26, esp. col. 7, ll. 63-66; col. 34,
ll. 1-31).

6 Genomic DNA is all the DNA sequences comprising the total genetic
information (genome) of a cell or organism. Since cDNA is DNA copied
from an mRNA by reverse transcription, cDNA lacks the introns present in
genomic DNA. See e.g., MOLECULAR CELL BIOLOGY, 5th ed., W.H.
Freeman and Company, New York, NY (2004), at G-3 and G-9.
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[21] Various control nucleic acid target elements may also be included on
the array, e.g. total genomic DNA or total genomic or cDNA from a
tissue with known abnormalities (Bao at col. 11, ll. 13-18).
[22] The first nucleic acid population comprises a mixture of mRNA or its
complementary cDNA, which is representative of gene expression in
the tissue sample and is labeled with a first marker, e.g., a red
fluorescent dye (Bao at col. 6, ll. 34-38; col. 17, ll. 5-9).
[23] The second nucleic acid population comprises a mixture of genomic
DNA which is representative of the tissue sample's total genomic, i.e.,
chromosomal, DNA and is labeled with a second marker, e.g., a green
fluorescent dye (Bao at col. 6, ll. 34-40; col. 17, ll. 5-9).
[24] The third nucleic acid population comprises a reference population
labeled with a third marker, e.g., an orange fluorescent dye (Bao at
col. 6, ll. 40-42; col. 17, ll. 5-9).
[25] The reference population may be total human genomic DNA from
normal tissue (Bao at col. 3, ll. 60-62; col. 6, ll. 46-49; col. 12, ll. 33-
36).
[26] The array of target elements is contacted, under hybridization
conditions, with the three labeled nucleic acid populations; the
presence and intensity of each marker is measured at each target
element; and, the ratios of the markers (e.g., first and third markers
and second and third markers) are determined for each target element
(Bao at col. 3, ll. 3-8 and 31-59; col. 6, ll. 22-55).
[27] Comparison of the ratios at a particular target element is said to
provide the copy number for the genomic DNA sequence and for
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cDNA sequences which are complementary to that target element
(Bao at col. 6, ll. 51-55).
C. Kuukasjärvi
[28] According to Kuukasjärvi, a major limitation in analyzing genetic
changes in the early stages of tumorigenesis is that the higher number
of normal cells in a tissue sample often mask genetic alterations in
premalignant and small malignant tumors (Kuukasjärvi at 94).
[29] While standard CGH7 allows genome-wide screening for DNA
sequence copy number abnormalities, it is said to require 0.5 to 1 µg
of genomic DNA, corresponding to roughly 50,000 to 100,000 diploid
cells from the tumor sample (Kuukasjärvi, ¶ bridging 94-95).
[30] Microdissected tumor samples are said to be too small to provide
enough DNA for standard CGH and, therefore, the DNA must be
amplified using PCR with degenerate probes (DOP-PCR) prior to
CGH analysis (Kuukasjärvi at 95).
[31] Kuukasjärvi describes an improved DOP-PCR method which includes
incorporating fluorescent labeled nucleotides directly into the PCR
reaction (Kuukasjärvi at 95).
[32] To test the sensitivity of DOP-PCR CGH, Kuukasjärvi made serial
dilutions of a known concentration of DNA extracted from the MCF-7
breast cancer cell line and used the dilutions as starting material in
DOP-PCR (Kuukasjärvi at 97).
[33] According to Kuukasjärvi, DOP-PCR could amplify template DNA
from concentrations as low as 25 pg DNA (Kuukasjärvi at 97).

7 Standard CGH uses chromosomes as the target elements for hybridization
(see e.g., Kuukasjärvi at 96 and Figure 3).
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[34] Further according to Kuukasjärvi, "CGH analysis showed that 50 pg
of DNA (corresponding roughly to two hypertetrapoloid MCF-7 cells)
was sufficient to produce copy number profiles, which were identical
to those obtained from a standard CGH protocol" (Kuukasjärvi at 97).
[35] PCR-based incorporation of fluorescent markers (labeled nucleotides)
was said "to be very effective and resulted in a high signal intensity in
CGH" (Kuukasjärvi at 100).
[36] Kuukasjärvi "concludes that CGH can now be efficiently used to
analyze DNA sequence gains and losses in small subpopulations of
cells from, e.g., premalignant and early lesions" (Kuukasjärvi at 100).
D. Rejections over the Prior Art and Rebuttal
[37] The Examiner finds that Bao teaches the method of claim 1 but for
use of dilution fractions of the second and third samples (Answer at 3-
4 and 6).
[38] Specifically, the Examiner finds that Bao teaches claim 1,
(a) contacting replicas of the array with a first
nucleic acid and a second nucleic acid sample . . . ,
wherein the first nucleic acid is labeled with a first
detectable label and the second sample with a second
detectable label and each comprise substantially
complete complements of the first genome (reference
nucleic acids) and second genome (cDNA sequences
complementary to expressed gene sequences) and
karyotype of first and second genome is known . . . ,
(b) contacting further replicas of the arrays with a
third sample (test or genomic nucleic acids or tumor
comprising substantially complete complement of
genomic nucleic acid of a third genome . . . [Answer at 3-
4, citations to Bao omitted, emphasis added.]

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[39] The Examiner finds that Kuukasjärvi teaches "the use of serial
dilutions of DNA in CGH analysis to increase the sensitivity of the
assay to detect the genetic variation in a dilution factor comprising a
single cell that aid in efficient detection of genetic variation in small
sub populations of cells . . ." (Answer at 6, citations to Kuukasjärvi
omitted).
[40] The Examiner concludes that it would have been obvious to modify
the method of Bao with a step of including dilution fractions of the
DNA sample as taught by Kuukasjärvi for the purpose of increasing
the sensitivity of the method down to a single cell fraction as taught
by Kuukasjärvi (Answer at 6).
[41] Appellant contends that the cDNA population disclosed by Bao does
not constitute a substantially complete genome because cDNAs do not
include introns and, therefore, Bao does not teach or suggest a method
using three populations of labeled probes comprising substantially
complete complements of genomes as defined by claim 1 (Br. at 6-7;
Reply Br.8 at 8-10).
[42] Appellant further contends that Bao only discloses (1) a mixture of
mRNA or its complementary cDNA and (2) a mixture of genomic
DNA, both of which are merely representative of (1) gene expression
in the tissue sample and (2) genomic status of the tissue sample,
respectively; and, neither is a substantially complete complement of
genomic nucleic acid (Reply Br. at 9).
[43] The Examiner responds that the scope of a “substantially” complete
genome or its complement was not defined in Appellant’s

8 Reply Brief filed 11 May 2007 (“Reply Br.”).
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specification and “could permit some variation in a genome resulting
in a broad range of percentage complementarity representing a
genome” (Answer at 9).
[44] According to the Examiner, “claim 1 is broad and can represent a
bacterial genome which do not contain intronic sequences, thus the
cDNA population [of Bao] does read on bacterial genome and the
assertions drawn to labeled cDNA genome” (Answer at 9).
[45] Appellant also argues that Kuukasjärvi teaches using dilutions to
obtain a suitable concentration of DNA to be used as a starting
material for DOP-PCR and, therefore, does not cure the deficiencies
of Bao (Br. at 7).
[46] The Examiner responds that the motivation in obviousness rejections
need not be the same as Appellant’s (Answer at 10).
[47] According to the Examiner, Kuukasjärvi teaches that serial dilutions
of the starting DNA template for PCR amplification shows that small
amounts of DNA could be used to accurately detect genetic
mosaicism in CGH assays and provides a reasonable expectation of
success of detecting genetic variation in each cell type of a cell
population (Answer at 10-11).
Other findings of fact are cited as necessary below.
III. Obviousness
A claimed invention is not patentable if the subject matter of the
claimed invention would have been obvious to a person having ordinary skill
in the art. 35 U.S.C. § 103(a); KSR Int'l Co. v. Teleflex, Inc., 127 S.Ct. 1727,
82 USPQ2d 1385 (2007); Graham v. John Deere Co. of Kansas City, 383
U.S. 1 (1966). Facts relevant to a determination of obviousness include (1)
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scope and content of the prior art, (2) any differences between the claimed
invention and the prior art, (3) the level of ordinary skill in the art, and (4)
relevant objective evidence of obviousness or non-obviousness. KSR, 127
S.Ct. at 1734, 82 USPQ2d at 1388; Graham, 383 U.S. at 17-18. All
limitations of claimed invention must be taught or suggested by the prior art
to establish prima facie obviousness. In re Royka, 490 F.2d 981, 985, 180
USPQ 580, 583 (CCPA 1974).
The dispositive issue here is whether Bao teaches or suggests a
method comprising contacting an array of nucleic acid target elements with
three populations of differentially labeled nucleic acid populations or
segments, each of which comprises a substantially complete complement of
its respective genome. Bao describes cohybridizing first, second and at least
one reference nucleic acid populations, each labeled with a different
fluorescent marker, to an array of nucleic acid target elements immobilized
on a solid substrate (FF 19). Bao teaches that the nucleic acid target
elements comprise total genomic DNA (FF 20). The first nucleic acid
population comprises a mixture of cDNA complementary to the mRNA
representative of gene expression in the tissue sample and is labeled with a
first marker, e.g., a red fluorescent dye (FF 22). The Examiner found that
this population of cDNA sequences reads on the second sample of step (a) of
claim 1 (FF 38). However, the method of claim 1, step (a) requires a second
sample comprising a plurality of genomic nucleic acid segments comprising
a substantially complete complement of the second genome. Since cDNA is
DNA copied from an mRNA by reverse transcription, cDNA lacks the
introns. Therefore, as argued by Appellant (FF 41), the cDNA population
used in Bao’s method cannot be genomic DNA because it lacks introns.
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Realizing this, the Examiner, for the first time,9 construes claim 1 as broad
enough to encompass a bacterial genome, which does not contain intronic
sequences (FF 44). However, Bao is directed to measuring gene expression
and chromosome abnormalities in the same tissue sample using array-based
CGH (FF 18). A unicellular bacterium cannot have multicellular tissues.
Hence, the Examiner's response is contradictory to the teachings of Bao.
Bao teaches a method using only two of the three genomic nucleic acid
samples required by the method of claim 1 (FF 22-24). Kuukasjärvi does
not cure the deficiencies of Bao. Since the Examiner has not established that
the prior art (Bao and Kuukasjärvi) teach or suggest all of the limitations of
claim 1, we will reverse the rejections of (i) claims 1-3, 5-7, 13-14, 18-25,
28-31, 34-41, 46, 54-58, 61-62 and 86 under § 103(a) as obvious over Bao
and Kuukasjärvi, and (ii) claims 47-49 under § 103(a) as obvious over Bao,
Kuukasjärvi and Bradley.
IV. Summary
In view of the record and for the reasons given, it is
ORDERED that the rejection of claims 1-3, 5-7, 13-14, 18-25, 28-31,
34-41, 46, 54-58, 61-62 and 86 under 35 U.S.C. § 103(a) as obvious over the
combined teachings of Bao and Kuukasjärvi is REVERSED;
FURTHER ORDERED that the rejection of claims 47-49 under 35
U.S.C. § 103 (a) as obvious over the combined teaches of Bao, Kuukasjärvi,
and Bradley is REVERSED; and

9 The Board’s review of the claims on appeal is not an independent analysis
in the first instance. The Examiner should set forth sufficient factual
findings and reasoning supporting claim construction in the first instance, to
permit a meaningful evaluation of the claimed invention vis-à-vis the
applied prior art.
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FURTHER ORDERED that this application is returned to the
Examiner for action consistent with the views espoused herein.
REVERSED

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