APPLICATION
`NUMBER
`61/798,925
`
`FILING or
`37l(c)DATE
`03/15/2013
`
`GRPART
`UNIT
`
`FIL FEE REC'D
`125
`
`86012
`VLP Law Group LLP
`555 Bryant Street
`Suite 820
`Palo Alto, CA 94301
`
`Ul\TfED STATES DEPA RTME'IT OF COMMERCE
`United States Patent and Trademark Office
`Adiliess. COMMISSIO'JER FOR PATENTS
`PO Box 1450
`Alexandria, Virgmia 22313-1450
`\VVi\V.USpto.gov
`
`ATTY.DOCKET.NO
`Sll-230PR1
`
`TOT CLAIMS IND CLAIMS
`
`CONFIRMATION NO. 6603
`FILING RECEIPT
`111111111111111111111111]~!1]!~1!~1!~1!111~ IIHHll lllll 111111111111111111
`
`Date Mailed: 05/14/2013
`
`Receipt is acknowledged of this provisional patent application. It will not be examined for patentability and will
`become abandoned not later than twelve months after its filing date. Any correspondence concerning the application
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`
`lnventor(s)
`
`Maximilian DIEHN, San Carlos, CA;
`Arash A. Alizadeh, San Mateo, CA;
`Aaron M. Newman, Palo Alto, CA;
`Scott V. Bratman, Palo Alto, CA;
`
`Applicant( s)
`
`Maximilian DIEHN, San Carlos, CA;
`Arash A. Alizadeh, San Mateo, CA;
`Aaron M. Newman, Palo Alto, CA;
`Scott V. Bratman, Palo Alto, CA;
`Power of Attorney:
`David Roise--47904
`
`If Required, Foreign Filing License Granted: 05/09/2013
`The country code and number of your priority application, to be used for filing abroad under the Paris Convention,
`is US 61 /798,925
`Projected Publication Date: None, application is not eligible for pre-grant publication
`Non-Publication Request: No
`Early Publication Request: No
`** SMALL ENTITY **
`Title
`
`IDENTIFICATION AND USE OF CIRCULATING TUMOR MARKERS
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`Statement under 37 CFR 1.55 or 1.78 for AIA (First Inventor to File) Transition Applications:
`page 1 of 3
`
`00001
`
`EX1025
`
`

`

`PROTECTING YOUR INVENTION OUTSIDE THE UNITED STATES
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`application under the Patent Cooperation Treaty (PCT). An international (PCT) application generally has the same
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`of patent applications on the same invention in member countries, but does not result in a grant of "an international
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`Almost every country has its own patent law, and a person desiring a patent in a particular country must make an
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`00002
`
`

`

`This license is to be retained by the licensee and may be used at any time on or after the effective date thereof unless
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`page 3 of 3
`
`00003
`
`

`

`1/19
`
`a
`
`·- .. ••, ·. ~ .·
`
`S11-230 PR1
`
`!:;:.:,-:>·n ..... OJ
`~ · +
`
`CAPP-Ser~
`se82>ctor
`::braiy
`
`C
`
`:'.-1u~nons µ-er paf.er~t dog2 sea~e;
`..... ~,--~ Training
`P--=(t{i5
`"'"*'' ... Va!~::'.:ation
`...... Ran.Qom
`
`Figure 1
`
`00004
`
`

`

`2/19
`
`e
`
`l :-:-:tx~ T ._;¼
`
`;.
`
`(~/~J.:·f)···S<:-:~J ~:t:1e-~;:~~r
`{-:...:njBf&d t>~: 9-2!n~:.:n1ic r~{~k;n}
`
`: ) •••••••••••••••••••••n•••••••••••••••••••••••••••••••••••••••••••n•
`
`f
`
`Figure 1 (cont.)
`
`00005
`
`

`

`3/19
`
`-
`
`Tissue
`biopsy
`
`Selector • ~
`
`Blood
`' d r aw
`<~
`
`Selector • •
`
`- I
`
`-
`
`d:~~~;ry
`
`J~ .. A z~·· ; .b. J~i..-:-~
`
`Fusion(s}?
`
`Enumerate
`base
`frequencies
`
`
`SNV/lnde!(s)'?
`
`I
`Fusion(s}?
`
`J discovery
`
`reporters
`
`~~~~:dc~I , - - . •
`t
`
`Adjust freq.?
`Input norm.
`
`r:ii~~;
`
`}--At.:: i .!:: ,'~A
`
`SNV/indel
`detection
`
`-
`
`Variant
`annotation
`
`Figure 2
`
`00006
`
`

`

`4/19
`
`b
`
`Known/suspected drivers
`Patienls/kb
`1
`10 1001000
`Kf/~,.;:;:;:;:;:;:;:;:;:;;:;:;:;:-
`PIK3CA :·:·:·:·:·:·:·:·:·:·:·:·
`CDKN2A :·:·:·:·:·:·:·:·:·:·:
`EGFR .,.,.,.,.,.,.,.,.,.,.
`KEAP1 ·:·:·:·:·:·:·:·:·:-:
`PTEN :-:·:·:·:·:·:·:·:-:
`BRAF :-:·:·:·:·:·:·:·:-:
`
`U)
`C:
`
`~ .....
`ci z
`
`0
`
`a
`
`2500
`
`2000
`
`1500
`
`fj'
`C:
`(I)
`::::,
`C"
`~ 1000
`LL
`
`500
`
`40000
`
`30000
`
`20000
`
`Known/suspected drivers
`Patients/ exon
`1
`10
`100
`
`PIK3CA ·=·=·=·=·=·=·=·=·=
`EGF
`·-:-----:---:----(cid:173)
`c~1:····:··:···,·:·····:··:···:·'f1'
`KEAP1 --:-----:---:----(cid:173)
`BRAF -:-:-:-:-:-:(cid:173)
`PTEN -:-:-:-:-
`
`I - ,
`50 510
`Recurrence Index (No. of patients per kb)
`
`8
`6
`No. of patients per exon
`
`f-,-
`10 105
`
`Figure 3
`
`00007
`
`

`

`a
`
`b
`
`C
`
`d
`
`e
`
`5/19
`
`Gene w reference
`
`Gene v reference
`
`Breakpoint identification
`
`Mate maps
`to gene v
`
`.;::: ·········3
`
`__ ... -···'' • •. • .. •. • ..... •. • ..... •. -~
`
`Male maps
`
`./
`
`~--, .. to gene w
`
`Breakpoint validation ___ /.-
`
`..•. ····
`
`>
`<
`Soft-clipped
`reads
`
`,
`:L.c.~ .. c...;,,.~
`.... <·-··•,,._ .. <•.Jim
`
`-..j..
`· Mapped gene w
`Soft-clipped
`R1 tit¢:itiscfATAoowrr:th t••ttt ??~
`Store all k-mers from
`mapped segment
`in hash table
`{e.g,, kz::4)
`
`Soft-clipped
`
`Mapped gene v
`
`TCTG
`GCTA
`JAGC
`
`Compare soft(cid:173)
`clipped segment
`to R1 using
`increments of
`size k
`
`Case 1a
`
`Possible orientations
`Caseza
`
`R1
`~::::::::::::;:;:;:;:;:;:;::::::::::::·
`
`Case 1b
`
`R1
`
`R2
`
`Case2b
`
`'""Case 2a
`
`Breakpoint adjustment
`
`Casez
`
`bp11.
`,j/':J
`R1 [/ t•t•?••t/t••••••?t/•••••t••••••
`
`A2 ~•~1111,,rnrnrnrn®
`bp2 y
`
`Breakpoint 2 "' bp2 + (x - y'J
`
`Breakpoint 2 = bp2 + (y ~ .x)
`
`Figure 4
`
`00008
`
`

`

`6/19
`
`a
`
`b
`
`ALK intron 19
`
`EML4 intron 13
`
`ROS1 intron 31
`
`SLC34A2 intron 4
`
`Figure 5
`
`00009
`
`

`

`7/19
`
`Phusion W!!h-Bead 32ng
`
`__ __.._ ___
`
`·:-~:.
`;-..~~
`''; <:~ .. ;.
`
`?~···
`
`,-..•. -.
`
`---------------~-
`
`-, .;:
`
`Phusion Star.card
`
`Phuslon Srar.dard 32ng
`
`i ---_,,,,,.
`
`a
`
`b
`
`C
`
`i
`
`0. "' E
`.,
`e
`
`C
`0
`
`"-
`
`los I
`{3 0.6 I
`I
`o 04 I
`I
`~ 0.2 I
`
`0
`
`Figure 6
`
`WGA Phusion W1!h·Bead
`
`KA.PA Wi!l'l·Bead 32ng
`
`'"'"''"
`l
`--~--
`
`......... ~t-:: ... ... :~:~-; ....... ;x: ...... ·~::;,
`>:, ,·· :,.:,.z:-,"-.,X·'
`
`KAPA With--Bead
`
`WGA Phusion With·Beaa
`
`•,; ·~ ... ~-
`
`• • • ' • • < • • • • • • ' • • <
`
`•
`
`•
`
`• <
`
`•
`
`' • • ' · -~ - · .~ : ·:
`
`!)_4 . . . . - - - - - - - - - - . .
`
`0.35
`
`><
`~ 0.3
`~ 0.25
`i 0.2
`<il
`~0.15
`5 0.1
`0.05
`
`()
`
`00010
`
`

`

`8/19
`
`P= 0.03
`
`b
`
`NS
`
`. .
`
`. .
`
`.P.:"' Cl.0l).4 ................. Ni;,
`
`......................... NS ...
`
`NS
`
`a
`
`1.6
`1.4
`
`-., 1.2
`I 1
`.,,
`.!!l 0.8
`.;
`~ 0.6
`2 0.4
`0.2
`
`0
`
`16hr/Cycle
`16hr116°C
`15min/20°C
`Adapter ligation duration and temperature
`
`50µL
`
`25µL
`Adapter ligation volume
`
`10µL
`
`d
`
`e
`
`f
`
`C
`
`2
`1.8
`1.6
`i 1.4
`·» al 1.2
`1
`.; e o.8
`~ 0.6
`0.4
`0.2
`0
`
`.!:!
`
`With-Bead
`Control
`SPRI bead processing
`
`100X
`10X
`Adapter:lragment molar ratio
`
`KAPA HiFi
`Phusion
`DNA polymerase used for PCR
`
`NuGEN
`KAPA
`Library Preparation Kit
`
`Figure 7
`
`00011
`
`

`

`9/19
`
`KAPA With-Bead 4ng
`
`KAPA With-Bead 32ng
`
`KAPA With-Bead 128ng
`
`---- nx·,,:J:,lr•
`
`KAPA With-Bead 4ng
`...............................................
`
`KAPA With-Bead 32ng
`
`KAPA With-Bead
`
`K>{•
`
`~e;.1
`
`:::¾
`;_,~,t.
`F~:~g,:,.:.~t L,•1"J• ·· ··/
`
`:,M
`
`;,~,)
`
`1St:
`
`.
`__::,x,
`Fta:J,~,€'H
`!'9!'>;r.~ -~~j
`
`~~---------~(,:,
`
`a
`
`b
`
`C
`
`cl
`QJ
`"-
`[ij-08
`E
`~ 0.6
`""
`~
`o 0.4
`
`C
`0
`i3 0.2
`~
`lL
`
`0
`
`I I
`
`Figure 8
`
`0.7
`
`1i5 0.4
`"'
`J;! 0.3
`
`0.1
`
`0
`
`0.6 ll a
`~ 0.5 I
`......•......
`······I······
`......•......
`......•......
`I
`0 0.2 I
`I
`I
`
`0.5
`
`@ 0.4
`1!
`·~ 0.3
`~
`.sc
`:§ 0.1
`
`~ 0.2 I
`
`0
`
`&4ng
`
`'832ng
`
`&128ng
`
`I
`
`00012
`
`

`

`a
`
`C
`
`e
`
`10/19
`
`b
`
`··'.'-:
`:-:_;.:·
`
`d
`
`<·=f · ... <:,,:.-' - - -
`
`~ 0·~.:::,":'":' ~ z:--tr~P} A -s.=:..:r·~~-.. +es:
`f-.i1E::(l~-:.~n .~~~--- ?5U'\ pt.:rcenij~e --*~-
`
`-:t-
`
`f·h.'::.:·~k~.i h>.;t r;;t !*<;Unern s<.:n1?..itk: n·~it-:rtion:s
`{:up 25 and t~nl~)n~ 2 (i 1(fl ~~Utd~;.:; *rs~~<.i)
`
`I
`I
`
`f
`
`,.
`1:·~·
`.-.;::.-
`
`G.n·:··
`ns)·
`-G. 77
`~!-~~i .
`G.t .. 7 . .:-·
`." ..................... w.·,., ............... ·.•.········· ........... .
`c,.4/--
`G .::;-r:~':°:'~:~:~~-~-::-.:-:~--::.-~':'::::~~-:-:::':::':':':-:':"::':"::'"::':"::':-:C:--:--:-:-:c:-:"':.-:-.':":':":".":
`
`-.. ,.. ...... ,._
`
`'.'.:~~
`
`i
`
`~~
`
`:1 4 5 f.;
`
`j B.
`
`r; H"! 11 1 :~ 1~1- -i'4
`
`Figure 9
`
`00013
`
`

`

`11 /19
`
`b
`
`10 ·
`
`; "'"''~'~ti""""' coaae.,_ -I
`
`6
`5
`4
`3
`2
`
`o·
`0
`
`..................... ; ......
`.. ~·
`
`2
`
`7
`6
`5
`4
`3
`Known fraction(%)
`
`8
`
`9 10
`
`Spike d
`
`C:
`
`"O
`
`~ g_..
`0 u
`i
`~
`'c
`~
`a.
`O"
`Q)
`{/J
`d.
`Cl.
`<(
`(.)
`
`a
`
`C
`
`1.0000
`
`.9995
`
`.9990
`
`.9985
`
`.9980
`
`~
`Cl)
`Ill
`:::,
`8
`iii
`~
`0
`:::,
`
`2
`
`4
`
`8 10 12 14 16 18 20 22 24
`6
`No. of reporters considered
`
`Mean correlation
`CV of 10% spike
`
`CV of 1 % spike
`······ CVof0.1% spike
`
`100
`
`10 ·
`
`R2 ,.0.995
`
`0.1
`
`Known fraction (%)
`
`II§!§! SNPs ~ lndel
`
`•
`
`Fusion
`
`~
`C:
`0
`l3
`:
`i
`¥ C.
`~ 0.1
`~
`
`C"
`
`2 4
`
`-
`
`1%
`0.1%
`6 8 10 12 14 16 18 20 22 24
`No. of reporters considered
`
`Figure 10
`
`00014
`
`

`

`12/19
`
`a
`
`C
`
`d
`
`4:D-- --,~,··
`,~ 0
`
`.... m
`.... -·-=··-.· ....... :.:.:-:,.·q-::..·.~
`~:._,,,.,,
`. ·.:.:::>:>-·.
`""<~:~,-~ .. ~--:.·.::,~:./··""""
`4(1
`-;?;)
`' '
`'
`~----..-------"-tl§' .... ·_· ----< - - - - , - - -~ - t )
`,,
`M~:,~~m
`
`. .;..0
`
`:?D'l·····················
`
`~1
`~ _..,....,. s lO
`x;,i
`~
`~ O"------
`
`... ~ .
`
`~ {.)cn~~1nt t:k~~:: EGf=f-1 L=&:~F{
`+· S~b(;S::.~s.~·- =EGFR 77~]t..-1
`
`-~·- [~~~~'{.t ~~: ~!~ t~~~ .. % (B.:.:3}
`
`Figure 11
`
`00015
`
`

`

`13/19
`13/19
`
`e
`
` x
`
`f
`
`g
`
`4
`
`SAng eearentp
`Pinas
`
`oO=
`
`Sa$.$
`
`SERN
`
`Figure 11 (cont.)
`Figure 11 (cont.)
`
`00016
`
`00016
`
`

`

`a
`
`b
`
`14/19
`
`EML4(chr2)
`
`,
`
`-
`
`.... _,,_,
`
`: x:..~}'K~·f·
`
`.
`
`...... -" ....•. =_-~-~--~-~"""I. :$irtz 1 !,
`1 $,p~ ~ ~~ ... :':.~
`L ~:~~ Jc-1:-·-~~_-~!_~_"=l~-=-'--_:"=~~~-~:t~~~-1
`...
`I :
`...,,. ..... ..,..,.,..,~-~t[~&.:.:.~~.;.·"':.: .. -....... ~ .... -.--.~~-~~-'""'Ei<on.,..::
`~~ .. :
`f-\~~~~~/:_:~·.1
`.... :: ... .:,:.,.M.....,
`,,,.,......,. ...... """'
`••• ,.. • ., ~,·,~~'"'-----Ex---::: ... :~~.,.:::~'""\"'/"
`L .• ?\s:i
`""'~
`I ...,.,
`
`ALK(chr2)
`
`!
`
`Predicted ALK Fusion Genes
`
`~ii~~j: ... ~h~
`•$<~~# 1····~~h#
`$oH 2~-ff ···~.M~
`;'·-·.· ---~
`: ... f:'..J
`
`E6;A20
`
`E13;A20
`
`E20;A20
`
`K24;A20
`
`R0S1 (chr6)
`
`Exon32
`
`Exon33
`
`Exon35
`
`Exon36
`
`Exon&
`
`-·-·.·e'W-f-.·.-·-
`
`Exon 34
`
`Exon7
`
`I ! ! Exon35
`Predicted R0S1 Fusion Genes
`(~iif.: {I Exon 32
`
`S4;R32
`
`S6;R34
`
`F2;R33
`
`R33;M7
`
`Figure 12
`
`S13del37;R34 u~~f-~.on34
`
`Exon 6 '~r- Exon 34
`
`Exon 33 -.:: r·-~ Exon 7
`
`00017
`
`

`

`15/19
`
`P= 0.03
`
`20
`
`15
`
`10
`
`5
`
`0
`
`•
`•
`t
`
`C)
`
`Smoking history
`9 Heavy
`e ught
`ONone
`
`(/)
`
`>
`z
`
`Cf)
`
`-
`
`0
`
`0 z
`
`Fusions
`absent
`
`Fusion(s)
`present
`
`Figure 13
`
`00018
`
`

`

`16/19
`
`Crizotinib initiated
`
`4 •
`
`2
`
`1
`
`P6
`
`1024
`
`~
`C .......
`p)
`:::J
`.......
`
`0 -0 z
`
`(0
`
`)>
`==a-
`----3
`-S
`
`~ ~
`
`>,
`()
`C
`(I)
`:::J
`CT
`(I)
`
`,.._ -(I)
`
`0.5
`
`(I)
`co
`+-'
`C
`$
`:::J
`~
`0.25
`
`-1
`1
`2
`4
`0
`3
`5
`6
`Months since initiation of therapy
`
`256
`
`-6)- SNV:TMEM132D
`-¢- SNV:TP53
`-0- SNV: NAV3 -ml- Fusion: KIF5B-ALK
`
`Figure 14
`
`00019
`
`

`

`17/19
`
`•, .·· . ~ .
`• '\1."' · ••
`
`Jr'.?:.~~~
`
`i
`ftxl.2
`l .... --.... ._--..,_._..,_._v..,._ .... ._._ .... ._ .... ._._ .... ._ .... ._v..-...-.,;,,,,.,._._ .... ._ .... ._Vl,. .... "9,.""'-"""",.Vl,. .... Vl,. ............ v..,-.....---"""
`
`.
`
`0
`
`I
`
`I
`
`0
`T""
`r---
`1-OIJ'?
`::::, lO ->
`iu
`CL CL
`(.) (.)
`:i...
`:i...
`Q) Q)
`CL CL
`I
`I
`T"" lO
`C"')o::t' cc
`(.) (.)
`
`Figure 15
`
`0
`
`EpCAM-APC
`
`00020
`
`

`

`18/19
`
`a 2 0 0 - - - - - - - - - - - - - - - -
`
`TCGA-05-4426
`TCGA-64-1680
`
`b
`
`Patient TCGA-05-4426
`
`150
`
`2 5: 100
`a::
`
`50
`
`0
`
`I
`
`C
`
`Remaining patients (n=161)
`Maximum
`- - Mean
`
`ROS1 exons
`
`Patient TCGA-64-1680
`
`. ~-·
`····················-s-.··-----------------------------
`... . .
`., ..
`·:::::::.·::.·::::.·::::.·:::·::.·:::·:::::::·::.·:::·:::::::::.·
`
`Discordant reads
`
`Read ID: B08PGABXX110404:3:2205:14145:64317
`
`ROS!
`
`CD74
`
`Figure 16
`
`00021
`
`

`

`19/19
`
`a
`
`(0) Pre-filter
`
`(1) Germline filter
`
`(2) cfDNA background filter ____. (3) Outlier detection
`
`1
`ob~·;_'~-~-~-~~ ~ o~t-
`
`1 300
`l 200
`i
`~ 100
`..,
`;{*"'~--
`'
`o+»·
`
`0
`
`20
`
`,..
`a
`
`"""
`I 300
`~ l 200
`i
`~ 100
`o'"l-
`
`0
`
`0
`
`l .;
`i
`~
`i
`0 e
`~
`af
`
`Robust Mattalanobis distanoe
`
`_____, llerative ocmelation
`
`20
`
`60
`40
`No. tags (deduped}
`
`80
`
`100
`
`60
`40
`No. tags (deduped)
`
`80
`
`100
`
`Cancer plasma cfDNA (P6)
`
`1 300
`l 200
`i
`
`~ 100
`
`1
`I
`i
`
`~
`
`0
`
`20
`
`eo
`40
`No. tags(deduped)
`
`80
`
`100
`
`0
`
`20
`
`60
`40
`No. tags {deduped)
`
`80
`
`100
`
`Robust Mahalanobls distance
`
`100
`
`C
`
`400
`
`60
`40
`No. tags (deduped}
`
`80
`
`,oo
`
`Post-Op plasma cfDNA (P1)
`
`pg
`
`0.6
`
`..
`
`RobustMahalanobis disiance
`
`__.., llerative correlation
`
`RobustMaha:anobisdistance
`
`............
`
`llerativecorrelation
`
`d
`
`P2
`
`" • f 0 I ~
`
`-"---
`
`--
`
`100
`50
`Robust M.ahalanobla distance
`
`~
`"
`I
`
`......._.
`
`ltarative correlai011
`
`Figure 17
`
`00022
`
`

`

`Doc Code: TR.PROV
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`Newman
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`00025
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`

`

`Patent Application
`Sll-230 PRl
`
`Identification and Use of Circulating Tumor Markers
`
`Statement of Governmental Support
`
`[0001] This invention was made with government support under grant number
`
`W81XWH- l 2- l -0285 awarded by the Department of Defense. The government has
`
`5
`
`certain rights in this invention.
`
`Background of the Invention
`
`[0002] Analysis of cancer-derived cell-free DNA (cfDNA) has the potential to
`
`revolutionize detection and monitoring of cancer. Noninvasive access to malignant
`
`DNA is particularly attractive for solid tumors, which cannot be repeatedly sampled
`
`10
`
`without invasive procedures. In non-small cell lung cancer (NSCLC), PCR-based
`
`assays have been used previously to detect recurrent point mutations in genes such as
`
`KRAS or EGFR in plasma DNA (Taniguchi et al. (2011) Clin. Cancer Res. 17:7808-
`
`7815; Gautschi et al. (2007) Cancer Lett. 254:265-273; Kuang et al. (2009) Clin.
`
`Cancer Res. 15:2630-2636; Rosell et al. (2009) N. Engl. J. Med. 361:958-967), but the
`
`15
`
`majority of patients lack mutations in these genes. Other studies have proposed
`
`identifying patient-specific chromosomal rearrangements in tumors via whole genome
`
`sequencing (WGS), followed by breakpoint qPCR from cfDNA (Leary et al. (2010)
`
`Sci. Transl. Med. 2:20ra14; McBride et al. (2010) Genes Chrom. Cancer 49: 1062-
`
`1069). While sensitive, such methods require optimization of molecular assays for
`
`20
`
`each patient, limiting their widespread clinical application. More recently, several
`
`groups have reported amplicon-based deep sequencing methods to detect cfDNA
`
`00026
`
`

`

`mutations in up to 6 recurrently mutated genes (Forshew et al. (2012) Sci. Transl. Med.
`
`4:136ral68; Narayan et al. (2012) Cancer Res. 72:3492-3498; Kinde et al. (2011) Proc.
`
`Natl Acad. Sci. USA 108:9530-9535). While powerful, these approaches are limited by
`
`the number of mutations that can be interrogated (Rachlin et al. (2005) BMC Genomics
`
`5
`
`6: 102) and the inability to detect genomic fusions.
`[0003] PCT International Patent Publication No. 2011/103236 desc1ibes methods for
`
`identifying personalized tumor markers in a cancer patient using "mate-paired"
`
`libraries. The methods are limited to monitoring somatic chromosomal rearrangements,
`
`however, and must be personalized for each patient, thus limiting their applicability and
`
`10
`
`increasing their cost.
`
`[0004] U.S. Patent Application Publication No. 2010/0041048 Al describes the
`
`quantitation of tumor-specific cell-free DNA in colorectal cancer patients using the
`
`"BEAMing" technique (Beads, Emulsion, Amplification, and Magnetics). While this
`
`technique provides high sensitivity and specificity, this method is for single mutations
`
`15
`
`and thus any given assay can only be applied to a subset of patients and/or requires
`
`patient-specific optimization. U.S. Patent Application Publication No. 2012/0183967
`
`Al describes additional methods to identify and quantify genetic variations, including
`
`the analysis of minor variants in a DNA population, using the "BEAMing" technique.
`
`[0005] U.S. Patent Application Publication No. 2012/0214678 Al describes methods
`
`20
`
`and compositions for detecting fetal nucleic acids and determining the fraction of cell(cid:173)
`
`free fetal nucleic acid circulating in a maternal sample. While sensitive, these methods
`
`analyze polymorphisms occurring between maternal and fetal nucleic acids rather than
`
`polymorphisms that result from somatic mutations in tumor cells. In addition, methods
`
`that detect fetal nucleic acids in maternal circulation require much less sensitivity than
`
`25
`
`methods that detect tumor nucleic acids in cancer patient circulation, because fetal
`
`nucleic acids are much more abundant than tumor nucleic acids.
`
`[0006] U.S. Patent Application Publication Nos. 2012/0237928 Al and
`
`2013/0034546 describe methods for determining copy number variations of a sequence
`
`of interest in a test sample comprising a mixture of nucleic acids. While potentially
`
`30
`
`applicable to the analysis of cancer, these methods are directed to measuring major
`
`structural changes in nucleic acids, such as translocations, deletions, and amplifications,
`
`rather than single nucleotide variations.
`
`- 2 -
`
`00027
`
`

`

`[0007] U.S. Patent Application Publication No. 2012/0264121 Al desclibes methods
`
`for estimating a genomic fraction, for example, a fetal fraction, from polymorphisms
`
`such as small base variations or insertions-deletions. These methods do not, however,
`
`make use of optimized libralies of polymorphisms, such as, for example, libraries
`
`5
`
`containing recurrently-mutated genomic regions.
`[0008] U.S. Patent Application Publication No. 2013/0024127 Al desclibes
`
`computer-implemented methods for calculating a percent contlibution of cell-free
`
`nucleic acids from a major source and a minor source in a mixed sample. The methods
`
`do not, however, provide any advantages in identifying or making use of optimized
`
`10
`
`libraries of polymorphisms in the analysis.
`
`[0009] PCT International Publication No. WO 2010/141955 A2 describes methods of
`
`detecting cancer by analyzing panels o