llIIlIlllIlIlIllIlIllIllIllIIlllIIlIllIlllIIlllIIllIlIlllIIllIllIIIllllIlIlIIIIIIIIIIIIIII
`US 20080090239Al
`
`&19) United States
`(12) Patent Application Publication
`SHOEMAKER et al.
`
`(10) Pub. No. : US 2008/0090239 A1
`Apr. 17, 2008
`(43) Pub. Date:
`
`(54) RARE CELL ANALYSIS USING SAMPLE
`SPLITTING AND DNA TAGS
`
`(76)
`
`Inventors: DANIEL SHOEMAKER, San Diego,
`CA (US); Martin Euchs, Uxbridge, MA
`(US); Neil X. Krueger, Roslindale, MA
`(US); Mehmet Toner, Wellesley Hills,
`MA (US); Darren Gray, Brookline,
`MA (US); Ravi Kapur, Stoughton, MA
`(US); Zihua Wang, Newton, MA (US)
`
`Correspondence Address:
`WILSON SONSINI GOODRICH 4 ROSATI
`650 PAGE MILL ROAD
`PALO ALTO, CA 94304-1050 (US)
`
`(21) Appl. No. :
`
`11/762, 747
`
`(22) Filed:
`
`Jun. 13, 2007
`
`Related U. S. Application Data
`
`(60) Provisional application No. 60/804, 819, filed on Jun.
`14, 2006. Provisional
`application No. 60/804, 817,
`filed on Jun. 14, 2006.
`
`Publication ClassiTication
`
`(51) Int. Cl.
`C12Q 1/20
`C12Q 1/68
`G01N 33/53
`(52) U. S. Cl.
`
`(2006. 01)
`(2006. 01)
`(2006. 01)
`. . . . . . . . . . . . . . . . . 435/6; 435/29; 435/7. 23
`
`(57)
`
`ABSTRACT
`
`to diagnose or prognose
`Described herein are methods
`cancer in a subject by enriching, detecting, and analyzing
`rare cells, e. g. , epithelial cells, in a sample from
`individual
`the subject. Also described are methods for labeling regions
`of genomic DNA in individual
`cells in said mixed sample
`labels wherein each label is specific to each
`with difierent
`the labeled regions of genomic DNA
`cell and quantifying
`from each cell in the mixed sample. More particularly
`the
`method includes detecting the presence of gene mutations
`in
`rare cells in a subsample.
`individual
`
`Average
`Hmv
`Direc Eon
`
`Lateral
`Flow
`Direction
`
`Ariosa Exhibit 1008, p. 1
`
`

`

`Patent Application Publication Apr. 17, 2008 Sheet 1 of 40
`
`US 2008/0090239 A1
`
`Average
`Hmv
`Direction
`
`Lateral
`Flow
`Direction
`
`Ariosa Exhibit 1008, p. 2
`
`

`

`Patent Application Publication Apr. 17, 2008 Sheet 2 of 40
`
`US 2008/0090239 A1
`
`Ariosa Exhibit 1008, p. 3
`
`

`

`Patent Application Publication Apr. 17, 2008 Sheet 3 of 40
`
`US 2008/0090239 A1
`
`Ariosa Exhibit 1008, p. 4
`
`

`

`Patent Application Publication Apr. 17, 2008 Sheet 4 of 40
`
`US 2008/0090239 A1
`
`g
`
`g
`
`9 O
`
`~Q g
`
`Q) 9
`
`Gg
`QQ
`
`Average
`Flew
`Dlrectloll
`
`Ariosa Exhibit 1008, p. 5
`
`

`

`Patent Application Publication Apr. 17, 2008 Sheet 5 of 40
`
`US 2008/0090239 A1
`
`'Owwlw Nw Xw XwLRwX
`
`4+ 5
`5 x~ 4 0 8 x+ 5 X+ 8 i+ 5 0
`x~ 8 x~ 8 0 & x& & x& R x& x
`
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`xy wg wy w&
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`
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`
`i&xi&Xi&wi&M«M iR, '
`00 00 x~wX x~0 w 5 w~
`
`%%%&%%%%%%%%XWV.
`
`chip
`
`Figure 1E
`
`Ariosa Exhibit 1008, p. 6
`
`

`

`Patent Application Publication Apr. 17, 2008 Sheet 6 of 40
`
`US 2008/0090239 A1
`
`Microposts and cells
`
`Figure 2A
`
`Antibody
`coGted
`posts
`
`Figure 2B
`
`Figure 2C
`
`Ariosa Exhibit 1008, p. 7
`
`

`

`Patent Application Publication Apr. 17, 2008 Sheet 7 of 40
`
`US 2008/0090239 A1
`
`Sample in
`
`Target cell
`
`Transferrin receptor l
`
`Magnetic flux field
`
`Polymer matrix with
`hard/soR magnetic material
`
`Silicon
`
`Supra or para magnetic
`par5de
`
`Monodonal antibody to
`hhnsferrin mepiN
`
`Figure 3
`
`Ariosa Exhibit 1008, p. 8
`
`

`

`Patent Application Publication Apr. 17, 2008 Sheet 8 of 40
`
`US 2008/0090239 A1
`
`Obtain sample
`
`400
`
`402
`
`404
`
`406
`
`408
`
`410
`
`NO
`
`412
`
`Stop
`
`414
`
`Enrich for rare
`cell
`
`Split enriched
`product
`
`Tag /amplify
`nucleic acids of
`interest
`
`Pool
`tagged/amplified
`nucleic acids
`
`Analyze
`pooled/tagged
`nucleic acids to
`detect allele
`abundance
`
`Identify wells
`with rare cells or
`rare DNA
`
`YES
`
`Determine
`condition of rare
`cells or DNA
`
`Figure 4
`
`Ariosa Exhibit 1008, p. 9
`
`

`

`Patent Application Publication Apr. 17, 2008 Sheet 9 of 40
`
`US 2008/0090239 A1
`
`Obtain sample
`
`500
`
`Enrich for rare
`cells
`
`Split enriched
`product
`
`Amplify highly
`polymorphic
`DNA regions
`
`Tag each
`polymorphic
`DNA region
`
`Pool
`amplicons
`
`Determine
`genotype of
`amplicons
`
`Analyze
`genotyping data
`
`502
`
`504
`
`506
`
`508
`
`510
`
`512
`
`514
`
`Identify cells with
`rare alleles
`
`NO
`
`Stop
`
`516
`
`Determine
`condition based
`on rare cell
`polymorphism
`
`Figure 5
`
`Ariosa Exhibit 1008, p. 10
`
`

`

`Patent Application Publication Apr. 17, 2008 Sheet 10 of 40
`
`US 2008/0090239 A1
`
`Into two or more
`
`discrete sites
`
`600
`
`602
`
`603
`
`Obtain sample from
`
`patient
`
`Enrich for rare cells
`
`Split enriched sample
`
`Perform molecular
`
`analysis test at each
`
`discrete site
`
`Determine condihon
`of patient
`
`605
`
`Figure 6
`
`Ariosa Exhibit 1008, p. 11
`
`

`

`Patent Application Publication Apr. 17, 2008 Sheet 11 of 40
`
`US 2008/0090239 A1
`
`2, ~ Fill-lsoly~ioit
`
`, 4. Exe~tttdsctlon
`
`H2
`
`3'
`
`rearrangement
`
`Molecular Inversion Probes are so named because the oligonucleotide probe central to the process undergoes a unimolecular
`from a molecule that cannot be amplified (step I), into a inolecule that can be amplified (step 6). This
`to genomic DNA (step 2) and an enzyinatic "gap fill" process that occurs in an
`is mediated by hybridization
`rearrangement
`allele-specific manner (step 3). The resulting circularized probe can be separated from cross-reacted or unreacted probes by a
`siruple exonuclease reaction (step 4). Figure 1 shows these steps.
`
`Figure 7A
`
`Ariosa Exhibit 1008, p. 12
`
`

`

`Patent Application Publication Apr. 17, 2008 Sheet 12 of 40
`
`US 2008/0090239 A1
`
`Well 41
`40 MIP probes
`
`fvvaavien
`
`avef label
`
`Well 42
`40 M P probes
`
`faaeavan
`
`eavl bbel
`
`POO I
`
`40 SNPs
`ags1-40
`
`40 SNPs
`Tags 4 t — 80
`
`ar0 OfOPrNXVfb
`
`Well 05QQ
`40 MIP probes
`
`fvavoaan
`
`~ e
`
`2Q, QQQ SNPs
`
`Hybridize
`
`vvl bbet
`
`40 ofoprfcoos
`
`40 SNPs
`Tags 19, 960 — 20, 000
`
`Figure 7B: 40-plex MIP assay performed for each of the 500 wells
`
`Ariosa Exhibit 1008, p. 13
`
`

`

`Patent Application Publication Apr. 17, 2008 Sheet 13 of 40
`
`US 2008/0090239 A1
`
`V
`r I
`5
`
`ID
`Ql
`Cl
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`55
`
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`
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`
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`CD
`
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`
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`
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`
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`13
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`
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`
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`
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`
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`
`Ql
`
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`E
`
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`
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`
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`C«CQ
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`'0
`
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`
`C5
`
`Ql
`
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`C!.
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`CD CLC 0
`O CI
`55 m. N
`CL ID
`
`CQ
`
`E
`
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`
`X
`
`CD
`
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`CI)
`ID
`
`CD
`Ql
`O
`O
`O
`0
`
`CQ
`
`IQ I
`Ql
`
`Ariosa Exhibit 1008, p. 14
`
`

`

`Patent Application Publication Apr. 17, 2008 Sheet 14 of 40
`
`US 2008/0090239 A1
`
`4-
`
`1536 well plate
`32 x 48 wells
`
`Current design has 14
`Increase to 16 or
`outputs.
`32 outpUts.
`Dispense into 32 wells
`simultaneously.
`
`Ariosa Exhibit 1008, p. 15
`
`

`

`Patent Application Publication Apr. 17, 2008 Sheet 15 of 40
`
`US 2008/0090239 A1
`
`Figure 8B
`
`Isometric View
`(not illustrative
`of actual well
`density)
`
`Figure 8C
`
`Cross-section
`
`I 0. 800
`
`O
`
`O. KO
`e OACO
`8
`u. 0. 2CO
`
`R obability Density Functions
`
`0
`
`200
`
`4m
`
`600
`
`800
`
`'i000
`
`1200
`
`hb of CTCs in sample
`
`Figure 9
`
`~ p(O) ~ P(~)
`p(2) ~ p(3)
`
`Probability of two
`CTCs in same well
`
`Ariosa Exhibit 1008, p. 16
`
`

`

`Patent Application Publication Apr. 17, 2008 Sheet 16 of 40
`
`US 2008/0090239 A1
`
`2AR
`
`A DISINTEGRIN
`
`Figure 10
`
`BONE SIALOPROTEIN (BSP)
`
`BREAST CANCER ESTROGEN-INDUCIBLE
`
`ACTIVATOR OF THYROID AND RFTINOIC
`
`SEQUENCE (BCEI)
`
`ACID RECEPTOR (ACTR)
`
`BREAST CANCER RESISTANCE PROTEIN
`
`CD82
`
`CD87
`
`CD9
`
`CEA
`
`ADAM 11
`
`(BCRP)
`
`ADIPOGENFSIS INHIBITORY FACTOR
`
`BREAST CANCER TYPE I (BRCAI)
`
`CELLULAR RETINOL-BINDING PROTEIN
`I (CRBPI)
`
`BREAST CANCER TYPE 2 (BRCA2)
`
`c-ERBB-2
`
`ALPHA 6 INTEGRIN SUBUNIT
`
`BREAST CARCINOMA AMPLIFIED
`
`ALPHA V INTEGRIN SUBUNIT
`
`ALPHA-CATENIN
`
`AMPLIFIED IN BREAST CANCER I (AIB I)
`
`AMPLIFIED IN BREAST CANCER 3 (AIB3)
`
`SEQUENCE 2 (BCAS2)
`
`CADHFRIN
`
`EPITI IELIAL CADHERIN-11
`
`CA DHERIN-ASSOCIATED PROTEIN
`
`AMPLIFIED IN BREAST CANCER 4 (AIB4)
`
`CALCITONIN RECEPTOR (CTR)
`
`AMYLOID PRECURSOR PROTEIN
`
`CALCIUM PLACENTAL PROTEIN (CAPL)
`
`SECRETASE (APPS)
`
`CALCYCLIN
`
`AP-2 GAMMA
`
`APPS
`
`ATP-BINDING CASSETTE TRANSPORTER
`
`(ABCT)
`
`CALLA
`
`CAM5
`
`CAPL
`
`CK7
`
`CK8
`
`CK18
`
`CK19
`
`CK20
`
`CLAUDIN-7
`
`c-MET
`
`COLLAGENASE
`
`FIBROBLAST
`
`COLLAGENASE
`
`INTERSTITIAL
`
`CARCINOEMBRYON IC ANTIGEN (CEA)
`
`COLLAGENASE-3
`
`PLACENTA-SPECIFIC (ABCP)
`
`ATP-BINDING CASSETTE SUBFAMILY C
`
`MEMBER (ABCC I)
`
`BAG-I
`
`BASIGIN (BSG)
`
`BC EI
`
`B-CELL DIFFERENTIATION FACTOR
`
`(BCDF)
`
`B-CELL LEUKEMIA 2 (BCL-2)
`
`B-CELL STIMULATORY FACTOR-2 (BSF-2)
`
`BC L-I
`
`BCL-2-ASSOCIATED X PROTEIN (BAX)
`
`BC RP
`
`BETA I INTEGRIN SUBUNIT
`
`BETA 3 INTEGRIN SUBUNIT
`
`BETA 5 INTEGRIN SUBUNIT
`
`BETA-2 INTERFERON
`
`BETA-CATENIN
`
`BETA-CATENIN
`
`CATENIN
`
`ALPHA I
`
`CATI IEPSIN B
`
`CATHEPSIN D
`
`CATHEPSIN K
`
`CATHEPSIN L2
`CATHEPSIN 0
`CATHEPSIN OI
`
`CATHEPSIN V
`
`CDIO
`
`CD146
`
`CD147
`
`CD24
`
`CD29
`
`CD44
`
`CD51
`
`CD54
`
`CD61
`
`CD66e
`
`COMMON ACUTE LYMPHOCYTIC
`
`LEUKEMIA ANTIGEN (CALLA)
`
`CONNEXIN 26 (Cx26)
`
`CONNEXIN 43 (Cx43)
`
`CORTACTIN
`
`COX-2
`
`CTLA-8
`
`CTR
`
`CTSD
`
`CYCLIN Dl
`
`CYCLOOXYGENASE-2
`
`CYTOKERATIN 18
`
`CYTOKERATIN 19
`
`CYTOKERATIN 8
`
`CYTOTOXIC T-LYMPHOCYTE-
`
`ASSOCIATED SERINE ESTERASE 8 (CTLA-
`
`8)
`
`DIFFERENTIATION-INI IIBITING
`ACTIVITY (D IA)
`
`Ariosa Exhibit 1008, p. 17
`
`

`

`Patent Application Publication Apr. 17, 2008 Sheet 17 of 40
`
`US2008/0090239A1
`
`DNA AMPLIFIED IN MAMMARY
`
`CARCINOMA
`
`I (DAM I)
`
`GAP JUNCTION PROTEIN ALPHA-1 (GJAI )
`GAP JUNCTION PROTEIN BETA-2 (GJB2)
`
`INTERLEUKIN-I ALPHA (IL-IA)
`
`INTERLEUKIN-I BETA (IL-IB)
`
`DNA TOPOISOMERASE ll ALPHA
`
`GCPI
`
`DR-NM23
`
`E-CADHERIN
`
`EMMPRIN
`
`EMSI
`
`GELATINASE A
`
`GELATINASE B
`
`GELATINASE (72 kDa)
`
`GELATINASE (92 kDa)
`
`ENDOTHELIAL CELL GROWTH FACTOR
`
`GLIOSTATIN
`
`INTERLEUKIN-11 (IL-11)
`
`INTERLEUKIN-17 (IL-17)
`
`INTERLEUKIN-1 8 (IL- I 8)
`
`INTERLEUKIN-6 (IL-6)
`
`INTERLEUKIN-8 (IL-8)
`
`INVERSELY CORRELATED WITH
`ESTROGEN RECEPTOR EXPRESSION-1
`(ICERE-I )
`KAII
`
`(ECGR)
`
`PLATELET-DERIVED (PD-ECGF)
`
`ENKEPHALINAS E
`
`EPIDERMAL GROWTH FACTOR
`RECEPTOR (EGFR)
`
`EPISIALIN
`
`GLUCOCORTICOID RECEPTOR
`INTERACTING PROTEIN I (GRIPI)
`
`GLUTATHIONE S-TRANSFERASE p
`GM-CSF
`
`GRANULOCYTE CHEMOTACTIC PROTEIN
`I (GCPI)
`
`KERATIN 18
`
`EPITHELIAL MEMBRANE ANTIGEN
`
`GRANULOCYTE-MACROPHAGF.
`
`-COLONY
`
`KERATIN 19
`
`(EMA)
`
`ER-ALPHA
`
`ERBB2
`
`ERBB4
`
`ER-BETA
`
`ERF-I
`
`STIMULATING FACTOR
`
`KISS-1
`
`GROWTH FACTOR RECEPTOR BOUND-7
`
`LEUKEMIA INHIBITORY FACTOR (LIF)
`
`(GRB-7)
`
`GSTp
`
`LIF
`
`LOST IN INFLAMMATORY BREAST
`
`HEAT-SHOCK COGNATE PROTEIN 70
`
`(HSC70)
`
`ERYTHROB3-POTENTIATING ACTIVITY
`
`(EPA)
`
`ESRI
`
`ESTROGEN RECEPTOR-ALPHA
`
`ESTROGEN RECEPTOR-BETA
`
`ETS-I
`
`EXTRACELLULAR MATRIX
`
`METALLOPROTEINASEINDUCER
`
`(EMMPRIN)
`
`FIB RON EC TIN RECEPTOR
`
`BETA POLYPEPTIDE (FNRB)
`
`FIBRONECTIN RECEPTOR BETA SUBUNIT
`
`FLK-I
`
`GAI S. 3
`
`GA733. 2
`
`GALECTIN-3
`
`GAMMA-CATENIN
`
`GAP JUNCTION PROTFIN (26 kDa)
`
`GAP JUNCTION PROTEIN (43 kDa)
`
`HEAT-STABLE ANTIGEN
`
`HEPATOCYTE GROWTH FACTOR (HGF)
`
`HEPATOCYTE GROWTH FACTOR
`
`RECEPTOR (HGFR)
`
`HEPATOCYTF. -STIMULATING FACTOR IH
`
`(HSF III)
`
`HER-2
`
`HER2/NEU
`
`HERMES ANTIGEN
`
`I IET
`
`HHM
`
`HUMORAL HYPERCALCEMIA OF
`
`MALIGNANCY
`
`(HHM)
`
`)CERE-I
`
`INT-I
`
`IN TERCELLULAR AD I I ES ION
`MOLECULE-1 (ICAM-I)
`
`INTERFERON-GAMMA-INDUCING
`
`FACTOR (IGIF)
`
`CANCER(LIBC)
`LOT (" LOST ON TRANSFORMATION" )
`LYMPHOCYTE HOMING RECEPTOR
`
`MACROPHAGE-COLONY STIMULATING
`
`FACTOR
`
`MAGF. -3
`
`MAMMAGLOBIN
`
`MC56
`
`M-CSF
`
`MDNCF
`
`MDR
`
`MELANOMA CELL ADHESION
`
`MOLECULE (MCAM)
`
`MEMBRANE METALLOENDOPEPTIDASE
`
`(MME)
`
`MEMBRANE-ASSOCIATED NEUTRAL
`
`ENDOPEPTIDASE (NEP)
`
`C Y STEIN E-RIC I I PROTEIN (MDC)
`
`METASTASIN (MTS-I)
`
`Ariosa Exhibit 1008, p. 18
`
`

`

`Patent Application Publication Apr. 17, 2008 Sheet 18 of 40
`
`US 2008/0090239 A1
`
`MLN64
`
`MMPI
`
`MMP2
`
`MMP3
`
`MMP7
`
`MMP9
`
`MMPI I
`
`MMP13
`
`MMP14
`
`MMP15
`
`MMP16
`
`MMP17
`
`MOESIN
`
`MONOCYTE ARGININE-SERPIN
`
`MONOCYTE-DERIVED NEUTROPHIL
`
`CHEMOTACTIC FACTOR
`
`MONOCYTE-DERIVED PLASMINOGEN
`
`ACTIVATOR INHIBITOR
`
`MTS-I
`
`MUC-I
`
`NUCLEAR RECEPTOR COACTIVATOR-I
`
`(NCoA-I )
`
`PLAKOGLOBIN
`
`NUCLEAR RECEPTOR COACTIVATOR-2
`
`(NCoA-2)
`
`NUCLEAR RECEPTOR COACTIVATOR-3
`
`PLASMINOGEN ACTIVATOR INHIBITOR
`(TYPE I)
`
`PLASMINOGEN ACTIVATOR INHIBITOR
`
`P1CoA-3)
`
`(TYPE 2)
`
`NUCLEOSIDE DIPHOSPHATE KINASE A
`
`PLASMINOGEN ACTIVATOR (TISSUE-
`
`(NDPKA)
`
`NUCLEOSIDE DIPHOSPHATE KINASE B
`
`(NDPKB)
`
`ONCOSTATIN M (OSM)
`
`TYPE)
`
`PLASMINOGEN ACTIVATOR
`
`(UROKINASE- TYPE)
`
`PLATELET GLYCOPROTEIN Illa (GP3A)
`
`ORNITHINE DECARBOXYLASE (ODC)
`
`PLAU
`
`OSTEOCLAST DIFFERENTIATION
`
`PLEOMORPI IIC ADENOMA GENE-LIKE I
`
`FACTOR (ODF)
`
`(PLAGLI)
`
`OSTEOCLAST DIFFERENTIATION
`
`POLYMORPHIC EPITHELIAL MUCIN
`
`FACTOR RECEPTOR (ODFR)
`
`OSTEONECTIN (OSN, ON)
`
`OSTEOPONTIN (OPN)
`
`OXYTOCIN RECEPTOR (OXTR)
`
`p27/kip1
`
`(PEM)
`
`PRAD I
`
`PROG ESTERONE RECEPTOR (PSR)
`
`PROGESTERONE RESISTANCE
`
`PROSTAGLANDIN ENDOPEROXIDE
`
`SYNTHASE-2
`
`MUC18
`
`MUCIN LIKE CANCER ASSOCIATED
`
`ANTIGEN (MCA)
`
`MUCIN
`
`MUC-I
`
`MULTIDRUG RESISTANCE PROTEIN I
`
`(MDR, MDR1)
`
`MULTIDRUG RESISTANCE RELATED
`PROTEIN-1 (MRP, MRP-I)
`
`N-CADHERIN
`
`NEP
`
`NEU
`
`NEUTRAL ENDOPEPTIDASE
`
`NEUTROPHIL-ACTIVATING PEPTIDE I
`(NAP I)
`
`NM23-Hl
`
`NM23-H2
`
`NMEI
`
`NME2
`
`p300/CBP COINTEGRATOR ASSOCIATE
`
`PROTEIN (p/CIP)
`
`p53
`
`p9Ka
`
`PAI-I
`
`PA I-2
`
`PARATHYROID ADENOMATOSIS
`
`I
`
`(PRADI)
`
`PARATHYROID HORMONE-LIKE
`
`HORMONE
`
`(PTHLH)
`
`PARATHYROIDHORMONE-RELATED
`
`PEPTIDE (PTHrp)
`
`P-CADHERIN
`
`PD-ECGF
`
`PDGF-U
`
`PROSTAGLANDIN G/H SYNTHASF;2
`
`PROSTAGLANDIN H SYNTHASE-2
`
`pS2
`
`PS6K
`
`PSORIASIN
`
`PTHLH
`
`PTHrP
`
`RAD51
`
`RAD52
`
`RAD54
`
`RAP46
`
`RECEPTOR-ASSOCIATED COACTIVATOR
`
`3 (RAC3)
`
`REPRESSOR OF ESTROGEN RECEPTOR
`
`PEANUT-REACTIVE URINARY MUCIN
`
`ACTIVITY (REA)
`
`(PUM)
`
`P-GLYCOPROTEIN (P-GP)
`
`PGP-I
`
`PHGS-2
`
`PHS-2
`
`SI OOA4
`
`S100A6
`
`S100A7
`
`S6K
`
`SART-I
`
`Ariosa Exhibit 1008, p. 19
`
`

`

`Patent Application Publication Apr. 17, 2008 Sheet 19 of 40
`
`US 2008/0090239 A1
`
`SCAFFOLD ATTACHMENT FACTOR B
`(SAF-B)
`
`TIMP3
`
`TIMP4
`
`UVOMORULIN
`
`VASCULAR ENDOTHELIAL GROWTH
`
`SCATTER FACTOR (SF)
`
`TISS UF. -TYPE PLASMINOGEN
`
`FACTOR
`
`SECRETED PHOSPHOPROTEIN-I
`
`(SPP-I)
`
`ACTIVATOR
`
`SECRETED PROTEIN
`
`ACIDIC AND RICH IN CYSTEINE (SPARC)
`
`TP53
`
`STANNICALC IN
`
`STEROID RECEPTOR COACTIVATOR-I
`(SRC-I )
`STEROID RECEPTOR COACTIVATOR-2
`(S RC-2)
`
`STEROID RECEPTOR COACTIVATOR-3
`
`(SRC-3)
`
`STEROID RECEPTOR RNA ACTIVATOR
`
`(SRA)
`
`STROMELYSIN-I
`
`STROMELYSIN-3
`
`TENASCIN-C (TN-C)
`
`TESTES-SPECIFIC PROTEASE 50
`
`THROMBOSPONDIN
`
`I
`
`THROMBOSPONDIN
`
`II
`
`THYMIDINE PHOSPHORYLASE (TP)
`
`THYROID HORMONE RECEPTOR
`
`ACTIVATOR MOLECULE 1 (TRAM-I)
`
`TRANSCRIPTIONAL INTERMEDIARY
`FACTOR 2 (TIF2)
`
`TREFOIL FACTOR I (TFFI)
`
`TSG101
`
`TSP-I
`
`TSPI
`
`TSP-2
`
`TSP2
`
`TSP50
`
`TUMOR CELL COLLAGENAS E
`STIMULATING FACTOR (TCSF)
`
`TUMOR-ASSOCIATED EPITHELIAL
`
`MUCIN
`
`uPA
`
`uPAR
`
`UROKINASE
`
`UROKINASE-TYPE PLASMINOGEN
`
`TIGHT JUNCTION PROTEIN I (TJPI)
`
`ACTIVATOR
`
`TIMP1
`
`TIMP2
`
`UROKINASE-TYPE PLASMINOGEN
`
`ACTIVATOR RECEPTOR (uPAR)
`
`VASCULAR ENDOTHELIAL GROWTH
`
`FACTOR RECEPTOR-2 (VEGFR2)
`
`VASCULAR ENDOTHELIAL GROWTH
`
`FACTOR-A
`
`VASCULAR PERMEABILITY FACTOR
`
`VEGFR2
`
`VERY LATE T-CELL ANTIGEN BETA
`
`(VLA-BETA)
`
`VIMENTIN
`
`VITRONECTIN RECEPTOR ALPHA
`
`POLYPEPTIDE (VNRA)
`
`VITRONECTIN RECEPTOR
`
`VON WILLEBRAND FACTOR
`
`VPF
`
`VWF
`
`WNT-I
`
`ZAC
`
`ZO-I
`
`ZON ULA OCCLUDENS-I
`
`Ariosa Exhibit 1008, p. 20
`
`

`

`Patent Application Publication Apr. 17, 2008 Sheet 20 of 40
`
`US 2008/0090239 A1
`
`Name
`
`NXK-ex18. 1(+)
`NXK-ex 18. 2(-)
`NXK-ex 18. 3(+)
`NXK-ex18. 4(-)
`NXK-ex19. 1(+)
`NXK-ex19. 2(-)
`NXK-ex19. 3(+)
`NXK-ex 19. 4(-)
`NXK-ex20. 1(+)
`NXK-ex20. 2(-)
`NXK-ex20. 3(+)
`NXK-ex20. 4(-)
`NXK-ex21. 1(+)
`NXK-ex21. 2(-)
`NXK-ex21. 3(+)
`NXK-ex21. 4(-)
`
`SEQ
`
`ID
`
`NO
`
`11
`
`12
`
`13
`
`14
`
`15
`
`17
`
`18
`
`19
`
`20
`
`21
`
`22
`
`23
`
`24
`
`25
`
`26
`
`Sequence (5' to 3')
`
`Exon
`
`Amplicon
`
`Size
`
`TCAGAGCCTGTGTTTCTACCAA
`
`TGGTCTCACAGGACCACTGATT
`
`TCCAAATGAGCTGGCAAGTG
`
`TCCCAAACACTCAGTGAAACAAA
`
`AAATAATCAGTGTGATTCGTGGAG
`
`GAGGCCAGTGCTGTCTCTAAGG
`
`GTGCATCGCTGGTAACATCC
`
`TGTGGAGATGAGCAGGGTCT
`
`ACTTCACAGCCCTGCGTAAAC
`
`ATGGGACAGGCACTGATTTGT
`
`ATCGCATTCATGCGTCTTCA
`
`ATCCCCATGGCAAACTCTTG
`
`GCAGCGGGTTACATCTTCTTTC
`
`CAGCTCTGGCTCACACTACCAG
`
`GCAGCGGGTTACATCTTCTTTC
`
`CATCCTCCCCTGCATGTGT
`
`Figure 11
`
`18
`
`18
`
`18
`
`19
`
`19
`
`19
`
`19
`
`20
`
`20
`
`20
`
`20
`
`21
`
`21
`
`21
`
`21
`
`534
`
`397
`
`495
`
`298
`
`555
`
`379
`
`526
`
`349
`
`Ariosa Exhibit 1008, p. 21
`
`

`

`Patent Application Publication Apr. 17, 2008 Sheet 21 of 40
`
`US 2008/0090239 A1
`
`Figure 12 A
`
`Figure 12 B
`
`Ariosa Exhibit 1008, p. 22
`
`

`

`Patent Application Publication Apr. 17, 2008 Sheet 22 of 40
`
`US 2008/0090239 A1
`
`Fi ure13A
`
`Fi ure 13B
`
`Fi ure 13C
`
`Figure 13D
`
`Figure 13E
`
`Figure 13F
`
`Figure 13A-F (Blue= nucleus, Red = X chromosome, Green = Y
`chromosome)
`
`Ariosa Exhibit 1008, p. 23
`
`

`

`Patent Application Publication Apr. 17, 2008 Sheet 23 of 40
`
`US 2008/0090239 A1
`
`Chromosome 21 Probe
`
`' X Probe
`
`Y Probe
`
`Figure 14
`
`Ariosa Exhibit 1008, p. 24
`
`

`

`Patent Application Publication Apr. 17, 2008 Sheet 24 of 40
`
`US 2008/0090239 A1
`
`Ariosa Exhibit 1008, p. 25
`
`

`

`Patent Application Publication Apr. 17, 2008 Sheet 25 of 40
`
`US 2008/0090239 A1
`
`Produd
`
`WBC HISTOGRAM
`
`RBC HISTOGRAM
`
`Waste
`
`50 100 150 200 250 300 350 400
`
`50
`
`100 150 200 250 XQ 350
`
`WBC HISTOGRAM
`
`RBC HISTOGRAM
`
`Blood
`
`WBC HISTOGRAM
`
`RBC HISTOGRAM
`
`Figure 16
`
`Ariosa Exhibit 1008, p. 26
`
`

`

`Patent Application Publication Apr. 17, 2008 Sheet 26 of 40
`
`US 2008/0090239 A1
`
`Ariosa Exhibit 1008, p. 27
`
`

`

`Patent Application Publication Apr. 17, 2008 Sheet 27 of 40
`
`US 2008/0090239 A1
`
`Ariosa Exhibit 1008, p. 28
`
`

`

`Patent Application Publication Apr. 17, 2008 Sheet 28 of 40
`
`US 2008/0090239 A1
`
`Inlet blood channel
`Inlet buffer channel
`
`ovcoctccc& hoch cc»»cclc
`C»»»»Z»CC»»»ha
`»»»»»»»»»h»h»»»!»»
`»»»»Z»»»»»»»»» Q C»»»»»!»»»»»»»h»hz!
`»»»»c»»»»»»el&»»»»»»»»c!'»»»l»»c»»
`»!»»»»»! 0»! 0 cl »»»» DQ QC! c ceo 0
`t&c»»»»*»»pc h»»e 'h»»c»»»»»»»»
`Cc&M»»&ehl&i »»cl&»»»»»»MC»»
`»»»»ccccccc o»»c»»ccc0 0 0
`
`Pest eecticn 0 gap size
`
`MM &44~
`
`~~~~~~8
`
`ggPa
`
`Pcs't Sectlcti 2 gap sue
`
`~~PPPAA
`t~~~~~~
`~~~~~sr&ûi@PXhl~
`X~»,
`
`» t
`
`Pest Sectien 5 gap size
`
`W»»(»»»h»l»»»» h»hn»»l
`lsroduct outlet channel
`
`Ariosa Exhibit 1008, p. 29
`
`

`

`Patent Application Publication Apr. 17, 2008 Sheet 29 of 40
`
`US 2008/0090239 A1
`
`let& dtrahrtet w~-+rdlerl
`Outlet chansrel mldth gwedamctj
`~hd et~eel midth
`ifwaete]f
`
`55
`dS
`56.
`
`59,
`
`Poet sectima 3
`rd parallel se&ions
`
`lkwhher
`
`Prcdtlct @em Size (ctrt elrt
`E&httated Flem rate, mlitt
`
`59/25
`55/18
`
`Ariosa Exhibit 1008, p. 30
`
`

`

`Patent Application Publication Apr. 17, 2008 Sheet 30 of 40
`
`US 2008/0090239 A1
`
`Clny dimensions: 66. 0 x 24. 9 nnn
`Post Field dimension: 51. 3 x 18. 9 mn
`Post diameter: 104 }nn
`Port dimensions
`Front side 2. 53 x2. 53 mm
`Back side 1. 66 x 1. 66 mm
`Snhstrafe: Silicon
`Etch depth: 100 pm
`
`Ariosa Exhibit 1008, p. 31
`
`

`

`Patent Application Publication Apr. 17, 2008 Sheet 31 of 40
`
`US 2008/0090239 A1
`
`RBC
`
`0
`
`2
`
`4
`
`20
`10
`6
`16
`22
`14
`12
`8
`Hydrodynamic Cell Diameter (pm)
`
`24
`
`Figure 20A
`
`RBC
`
`P/ately
`
`0
`
`2
`
`4
`
`%BC
`
`CTC
`
`16
`6
`10
`14
`20
`12
`22
`8
`Hydrodynamic Cell Diameter (pm)
`
`24
`
`Ariosa Exhibit 1008, p. 32
`
`

`

`Patent Application Publication Apr. 17, 2008 Sheet 32 of 40
`
`US 2008/0090239 A1
`
`RBC
`
`Platal
`
`0
`
`2
`
`4
`
`Figure 20C
`
`RBC
`
`Platel
`
`Size Cutoff
`
`WBC
`
`CTC
`
`J4
`16
`6
`20
`10
`22
`8
`12
`Hydrodynamic Cell Diameter {pm)
`
`24
`
`Size Cutoff
`
`Population of cells
`indicative of a
`(lisease state
`
`&BC
`
`0
`
`2
`
`4
`
`6
`16
`10
`20
`22
`14
`12
`8
`Hydrodynamic Cell Diameter {pm)
`
`24
`
`Figure 20D
`
`Ariosa Exhibit 1008, p. 33
`
`

`

`Patent Application Publication Apr. 17, 2008 Sheet 33 of 40
`
`US 2008/0090239 A1
`
`Ariosa Exhibit 1008, p. 34
`
`

`

`Patent Application Publication Apr. 17, 2008 Sheet 34 of 40
`
`US 2008/0090239 A1
`
`10s cells
`
`164 cells
`
`102 cells
`
`Result
`
`Enrichment
`
`Capture
`
`Molecular
`Analysis
`
`EQI%5
`
`yyT
`h15bp
`
`Molecular
`Infortttatlon
`
`Blood
`draw
`
`CTCs and
`some WBCs
`
`CTCs
`
`Figure 22
`
`First Strand Synthesis
`
`Reverse Transariptase
`
`PCR empliTieatten 4tt
`
`Template: 10'l» af eDNA reaettan
`
`PCR ampllttaattpntt2
`
`) Template: 4y»otPCR ttt
`
`8 I3 hp
`
`~ M&z
`
`242 &p
`
`Ariosa Exhibit 1008, p. 35
`
`

`

`Patent Application Publication Apr. 17, 2008 Sheet 35 of 40
`
`US 2008/0090239 A1
`
`Ariosa Exhibit 1008, p. 36
`
`

`

`Patent Application Publication Apr. 17, 2008 Sheet 36 of 40
`
`US 2008/0090239 A1
`
`TCA, , OCATTTTGGOC9:G8„C, A'GP, C„t;GET. 'l39GP, GT FUGATA
`""'
`sz
`
`54o
`
`Erg
`
`09C
`
`(i
`
`~(v„
`
`CAA + CAG (ailent) at 2361
`ACG + ATG (T79()M) at 2369
`
`CTG W CGG (L828Ft) at 2573
`
`ACC W ACT (ailant) at 2709
`
`Ariosa Exhibit 1008, p. 37
`
`

`

`Patent Application Publication Apr. 17, 2008 Sheet 37 of 40
`
`US 2008/0090239 A1
`
`&BC cOMA
`
`H1850 cDMA
`
`Target rnRNA
`
`Figurc 26A
`
`EGFR
`
`Tissue
`1 Renel Blood Vessel
`2 Bload Monanualeer Cells (PBMC)
`3 Colon
`
`26, 916
`24. CQ
`8
`WG
`24. 28 21, 968
`24. 49 19, 224
`
`Ariosa Exhibit 1008, p. 38
`
`

`

`Patent Application Publication Apr. 17, 2008 Sheet 38 of 40
`
`US 2008/0090239 A1
`
`PCR @1
`N IH-373 — 100 pg total RNA = -2G cells
`NCl-H1975 — 160 pg total RNA = -30 cells
`COS-T — 160 pg total RNA = -6 cells
`
`Ariosa Exhibit 1008, p. 39
`
`

`

`Patent Application Publication Apr. 17, 2008 Sheet 39 of 40
`
`US 2008/0090239 A1
`
`1000 ng FBME RNA &no eorms&
`1 ng H1975 plus
`PBMC RNA
`
`1 ng l-l1 975 alone
`200 Ag H19HRllA inn nBWL Rg
`
`tgaRNS)
`
`100 1000 10000 100900
`
`H~O CmgOI
`
`813 bp
`
`I'ice 28A
`
`'! ng H1875 plus:
`
`M
`m
`
`~
`
`N
`
`m
`
`m
`
`s
`
`Spurious PBMC
`arnplicons
`visible at high
`dilution
`
`813 bp
`
`TT~~ TIBte: 2 1II
`(40iu) Of the
`gnrmsponding
`PCR Rgggtion
`
`1
`
`Specific arTipiicon
`produced even et
`1QQQ:1 difution
`
`Ariosa Exhibit 1008, p. 40
`
`

`

`Patent Application Publication Apr. 17, 2008 Sheet 40 of 40
`
`US 2008/0090239 A1
`
`EGFR WT (138 bp)
`EGFM15bp
`(123 bp)
`
`Ariosa Exhibit 1008, p. 41
`
`

`

`US 2008/0090239 A1
`
`Apr. 17, 2008
`
`RARE CELL ANALYSIS USING SAMPLE
`SPLITTING AND DNA TAGS
`
`CROSS REFERENCE TO RELATED
`APPLICATIONS
`[0001] This application claims priority, under 35 U. S. C.
`)119, to U. S. provisional patent application Nos. 60/804, 819
`and 60/804, 817 both filed on Jun. 14, 2006 and incorporated
`herein by reference
`in their entirety.
`
`BACKGROUND OF THE INVENTION
`
`[0002] Analysis of specific cells can give insight
`into a
`variety of diseases. These analyses can provide non-invasive
`tests for detection, diagnosis and prognosis of diseases such
`the risk of
`as cancer or fetal disorders,
`thereby eliminating
`invasive diagnosis. Regarding
`fetal disorders, current pre-
`and chorionic villus
`natal diagnosis, such as amniocentesis
`(CVS), are potentially harmful
`to the mother and
`sampling
`to the fetus. The rate of miscarriage
`for pregnant women
`is increased by 0. 5-1%, and that
`amniocentesis
`undergoing
`is slightly higher for CVS. Because of the inherent
`figure
`risks posed by amniocentesis and CVS, these procedures are
`to older women, e. g. , those over 35 years
`ofiered primarily
`of age, who have a statistically greater probability of bearing
`children with congenital defects. As a result, a pregnant
`woman at the age of 35 has to balance an average risk of
`0. 5-1% for inducing an abortion by amniocentesis against an
`for trisomy 21 of less than 03%.
`age related probability
`[0003] Regarding prenatal diagnostics, some non-invasive
`to screen for fetuses
`methods have already been developed
`risk of having
`specific congenital defects. For
`at higher
`and levels of
`serum alpha-fetoprotein,
`example, maternal
`unconjugated estriol and human chorionic gonadotropin can
`be used to identify a proportion of fetuses with Down' s
`these
`false
`tests sufier from many
`syndrome. However,
`positives. Similarly, ultrasonography
`is used to determine
`congenital defects involving neural
`tube defects and limb
`abnormalities, but such methods are limited to time periods
`fifteen weeks of gestation
`after
`and present unreliable
`results.
`the blood of
`[0004] The presence of fetal cells within
`to develop a prenatal
`pregnant women ofiers the opportunity
`and thereby elimi-
`diagnostic
`that replaces amniocentesis
`nates the risk of today's invasive diagnostics. However, fetal
`cells represent a small number of cells against
`the back-
`ground of a large number of maternal cells in the blood
`which make the analysis time consuming and prone to error.
`
`[0005] With respect to cancer diagnosis, early detection is
`of paramount
`importance. Cancer is a disease marked by the
`uncontrolled proliferation of abnormal cells. In normal
`tis-
`sue, cells divide and organize within the tissue in response
`cells. Cancer cells do not
`to signals
`from surrounding
`in the same way to these signals, causing them to
`respond
`proliferate and, in many organs, form a tumor. As the growth
`of a tumor continues, genetic alterations may accumulate,
`as a more aggressive growth phenotype of the
`manifesting
`cancer cells. If left untreated, metastasis,
`the spread of
`cancer cells to distant areas of the body by way of the lymph
`system or bloodstream, may ensue. Metastasis results in the
`formation of secondary
`tumors at multiple sites, damaging
`healthy tissue. Most cancer death is caused by such second-
`ary tumors. Despite decades of advances in cancer diagnosis
`
`and therapy, many cancers continue
`to go undetected until
`late in their development. As one example, most early-stage
`lung cancers are asymptomatic
`and are not detected in time
`for curative
`five-year
`treatment,
`in an overall
`resulting
`survival rate for patients with lung cancer of less than 15%.
`in which lung cancer is detected
`in those instances
`However,
`is much more
`and treated at an early stage, the prognosis
`favorable.
`[0006] The methods of the present invention allow for the
`detection of fetal cells and fetal abnormalities when fetal
`cells are mixed with a population of maternal cells, even
`when the maternal cells dominate
`the mixture. In addition,
`the methods of the present
`invention can also be utilized to
`detect, diagnose, or prognose cancer.
`
`SUMMARY OF THE INVENTION
`
`[0007] The present
`for the
`to methods
`relates
`invention
`detection of fetal cells or cancer cells in a mixed sample. In
`the present invention provides methods for
`one embodiment,
`fetal abnormalities
`in a sample comprising
`fetal
`determining
`cells that are mixed with a population of maternal cells. In
`the presence of fetal cells
`some embodiments,
`determining
`labeling one or more
`comprises
`and fetal abnormalities
`regions of genomic DNA in each cell from a mixed sample
`least one fetal cell with difi'erent
`labels
`at
`comprising
`wherein each label is specific to each cell. In some embodi-
`the genomic DNA to be labeled comprises one or
`ments,
`particularly STRs or SNPs
`more polymorphisms,
`the methods of the inven-
`[0008]
`In some embodiments,
`detecting the presence of fetal
`tion allow for simultaneously
`cells and fetal abnormalities when fetal cells are mixed with
`a population of maternal cells, even when the maternal cells
`the mixture. In some embodiments,
`the sample is
`dominate
`enriched to contain at least one fetal and one non fetal cell,
`the cells of the enriched popu-
`and in other embodiments,
`lation can be divided between two or more discrete locations
`locations. Examples of
`that can be used as addressable
`locations
`include wells, bins, sieves, pores,
`addressable
`geometric sites, matrixes, membranes, electric traps, gaps or
`obstacles.
`[0009] In some embodiments,
`the methods comprise label-
`ing one or more regions of genomic DNA in each cell in the
`labels, wherein each label is
`enriched sample with difi'erent
`to each cell, and quantifying
`labeled DNA
`the
`specific
`regions. The labeling methods can comprise adding a unique
`for each cell in the mixed sample. In some
`tag sequence
`the pres-
`embodiments,
`the unique
`identifies
`tag sequence
`ence or absence of a DNA polymorphism
`in each cell from
`the mixed sample. Labels are added to the cells/DNA using
`reaction, which can be performed by PCR
`an amplification
`methods. For example, amplification
`can be achieved by
`multiplex PCR. In some embodiments, a further PCR ampli-
`fication is performed using nested primers for the genomic
`DNA region(s).
`[0010] In some embodiments,
`the DNA regions can be
`amplified prior to being quantified. The labeled DNA can be
`in some
`sequencing methods, which,
`quantified
`using
`the DNA regions. The
`embodiments, can precede amplifying
`amplified DNA region(s) can be analyzed by sequencing
`methods. For example, ultra deep sequencing can be used to
`provide an accurate and quantitative measurement of the
`for each STR or SNP. In other embodi-
`allele abundances
`
`Ariosa Exhibit 1008, p. 42
`
`

`

`US 2008/0090239 A1
`
`Apr. 17, 2008
`
`can be used to declare the
`ments, quantitative
`genotyping
`presence of fetal cells and to determine
`the copy numbers of
`the fetal chromosomes. Preferably, quantitative genotyping
`is performed using molecular
`inversion probes.
`[0011] The invention also relates to methods of identifying
`cells from a mixed sample with non-maternal genomic DNA
`said cells with non-maternal genomic DNA
`and identifying
`the ratio of maternal
`as fetal cells. In some embodiments,
`to
`paternal alleles is compared on the identified fetal cells in the
`mixed sample.
`[0012] In one embodiment,
`for a
`the invention provides
`method for determining
`a fetal abnormality
`in a maternal
`that comprises at least one fetal and one non fetal
`sample
`cell. The sample can be enriched to contain at least one fetal
`sample can be arrayed
`cell, and the enriched maternal
`into
`a plurality of discrete sites. In some embodiments,
`each
`discrete site comprises no more than one cell.
`[0013] In some embodiments,
`comprises
`the
`invention
`labeling one or more regions of genomic DNA from the
`that are specific to each DNA
`arrayed samples using primers
`region or location,
`the DNA region(s), and
`amplifying
`the labeled DNA region. The labeling of the
`quantifying
`labeling each region with a
`DNA region(s) can comprise
`tag sequence, which can be used to identify
`the
`unique
`presence or absence of a DNA polymorphism
`on arrayed
`cells and the distinct location of the cells.
`[0014] The step of determining
`can comprise
`identifying
`locations, which can
`non-maternal
`alleles at the distinct
`the ratio of maternal
`to paternal
`from comparing
`result
`the method of
`alleles at the location. In some embodiments,
`in an arrayed sample can
`a fetal abnormality
`identifying
`the genomic DNA regions. The
`further comprise amplifying
`genomic DNA regions can comprise one or more polymor-
`phisms, e. g. , STRs and SNPs, which can be amplified using
`including multiplex PCR. An additional
`PCR methods
`step can be performed using nested primers.
`amplification
`[0015] The amplified DNA region(s) can be analyzed by
`sequencing methods. For example, ultra deep sequencing
`can be used to provide an accurate and quantitative mea-
`surement of the allele abundances
`for each STR or SNP. In
`quantitative genotyping can be used to
`other embodiments,
`declare the presence of fetal cells and to determine
`the copy
`numbers of the fetal chromosomes. Preferably, quantitative
`is performed using molecular
`inversion probes.
`genotyping
`[0016] In one embodiment,
`the invention provides meth-
`ods for diagnosing
`a cancer and giving a prognosis by
`obtaining and enriching a blood sample from a patient for
`into discrete
`epithelial cells, splitting
`the enriched sample
`locations, and performing one or more molecular
`and/or
`analyses on the enriched and split sample.
`morphological
`the level of
`The molecular analyses can include detecting
`expression or a mutation of gene disclosed
`in FIG. 10.
`comprises performing molecular
`the method
`Preferably,
`analyses on EGFR, EpCAM, GA733-2, MUC-I, HER-2, or
`in each arrayed cell. The morphological
`Claudin-7
`analyses
`can include
`and/or characterizing
`identifying,
`quantifying
`mitochondrial DNA, telomerase, or nuclear matrix proteins.
`In some embodiments, morphological
`include
`analyses
`the stained rare cells using
`rare cells and imagin