PATENT
`Attorney Docket No.: EXCT-31920/US-3/CON
`
`IN THE UNITED STATES PATENT AND TRADEMARKOFFICE
`
`In re Application of:
`Serial No.:
`Filed:
`Entitled:
`
`Janelle J. Bruinsma
`14/145,082
`12/31/2013
`ISOLATION OF NUCLEIC ACID
`
`Confirmation No.:
`Group No.:
`Examiner:
`
`9571
`1637
`AaronPriest
`
`AMENDMENT UNDER37 C.F.R. §1.116 AFTER
`FINAL OFFICE ACTION MAILED JULY 6, 2015
`AND
`REQUEST FOR AFTER-FINAL CONSIDERATION UNDER AFCP2.0
`
`VIA EFS WEB
`Commissionerfor Patents
`P.O. Box 1450
`Alexandria, Virginia 22313-1450
`
`CERTIFICATE: OF- EFS WEB TRANSMISSION: UNDER 37-C.F.R.§ 1.8
`
`L-hereby. certify that. this correspondence (along swith any referred to. as. being attached: or enclosed):is, onthe date
`shown: below; bein: transmitted ‘tothe United: States :Patent:and “Trademark: Office: transmitted via the: Office electronic
`filing system in-accordance with 37: C.F. R.§ b6ta)(4):
`
`Mary: Ann:D: Brow, Reg: No. 42,363
`
`Dated:
`
`July: 3422015
`
`By:
`
`/Mary Ann}. Brow/.
`
`This communication is an After Final Amendment under 37 C.F.R. §1.116, responsive to
`
`the Final Office Communication mailed July 6, 2015, with a response due on or before October
`
`6, 2015, and is filed with a Request for After-Final Consideration under AFCP 2.0. Applicants
`
`respectfully request entry of this amendment.
`
`The Commissioneris hereby authorized to charge any fees during the entire pendency of
`
`this application, including fees due under 37 C.F.R. §§ 1.16 and 1.17 that may be required,
`
`including any required extension of time fees, or credit any overpayment to Deposit Account 50-
`
`4302, referencing Attorney Docket No.: EXCT-31920/US-3/CON. This paragraphis intended to
`
`be a CONSTRUCTIVE PETITION FOR EXTENSION OF TIMEin accordance with 37 C.F.R.
`
`§ 1.136(a)(3).
`
`Amendments to the Claimsare reflected in the Listing of Claims beginning on page 2.
`
`Remarksbegin on page5.
`
`Geneoscopy Exhibit 1029, Page 1
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`Geneoscopy Exhibit 1029, Page 1
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`

`

`PATENT
`Attorney Docket No.: EXCT-31920/US-3/CON
`
`AMENDMENTS TO THE CLAIMS:
`
`Thislisting of the claims will replace all prior listings and versions of claims in the
`
`application:
`
`1.
`
`(currently amended) A system for isolating a target human DNA from a humanstool
`
`sample, the system comprising:
`
`a)
`
`b)
`
`c)
`
`a volumeof stool homogenization solution suitable for processing a human
`
`stool sample having a massofat least 4 grams;
`
`an inhibitor-adsorbing composition comprising insoluble
`
`polyvinylpolypyrrolidoneparticles;
`
`a spin filter, comprising
`
`1)
`
`ll)
`
`iil)
`
`a hollow body;
`
`a bottom end; and
`
`an open top end opposite the bottom end,
`
`wherein the hollow bodyofsaid spin filter is made from a porousfiltering material;
`
`d)
`
`guanidine thiocyanate; and
`
`
`
`
`
`actin, a plurality of different magnetic target-specific capture particles, wherein
`
`different target-specific capture particles comprise covalently-attached
`
`oligonucleotides complementary to at least a portion of different target human
`
`DNAs, and wherein said different target human DNAs comprise NDRG4, BMP3,
`
`and KRAS.
`
`2.
`
`(original) The system of claim 1, further comprising a magnet.
`
`3-4.
`
`(cancelled)
`
`Geneoscopy Exhibit 1029, Page 2
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`Geneoscopy Exhibit 1029, Page 2
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`

`

`PATENT
`Attorney Docket No.: EXCT-31920/US-3/CON
`
`(previously presented) The system of claim 1 wherein said polyvinylpolypyrrolidoneis
`
`in a premeasured form.
`
`(previously presented) The system of claim 3 wherein said polyvinylpolypyrrolidoneis
`
`provided as a capsule or pressed tablet comprising a premeasured amount of
`
`polyvinylpolypyrrolidone.
`
`(cancelled)
`
`(previously presented) The system of claim 1, further comprising an elution solution
`
`and/or a washsolution.
`
`9-12.
`
`(cancelled)
`
`13.
`
`(previously presented) The system of claim 1, further comprising a sample container
`
`configured to hold said stool sample.
`
`14.
`
`(original) The system of claim 1 further comprising a vessel in whichto hold isolated
`
`target human DNA.
`
`15.
`
`(original) The system of claim 1, further comprising a shipping container.
`
`16.
`
`(original) The system of claim 1, further comprising a control reagent.
`
`17.
`
`(original) The system of claim 16, wherein said control comprises a nucleic acid.
`
`18-23. (cancelled)
`
`24.
`
`(currently amended) The system of claim 1, wherein said covalently-attached
`
`oligonucleotide comprises a sequenceselected from the group consisting of SEQ ID
`
`NOS:+52-3 and 6-7.
`
`Geneoscopy Exhibit 1029, Page 3
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`Geneoscopy Exhibit 1029, Page 3
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`

`

`PATENT
`Attorney Docket No.: EXCT-31920/US-3/CON
`
`25.
`
`(previously presented) The system of claim 1, further comprising a magnetic target-
`
`specific capture particle comprising an oligonucleotide that is complementary to at least a
`
`portion of a humanreference DNA.
`
`(currently amended) The system of claim 1, comprising-a-phiraltyefdifferentimagnetic
`26.
`
`
`
`
`
`differentiarcethumanDNAsandwherein saiddifferenttargethumanDNAs-comprise2
`
`
`
`
`
`plurality of target haman DNAsseleeted-from+the-eroup-consistineoffurther comprises
`
`
`vimentin,NDRG4,BMPS,TFPI2, or beta-actin-andiAs.
`
`27.
`
`(previously presented) The system of claim 26, wherein said covalently-attached
`
`oligonucleotides comprise sequences selected from the group consisting of SEQ ID
`
`NOS:1-7.
`
`28.
`
`(currently amended) The system of claim 2625, wherein said-phiraht-efdifferenttarget
`
`humanDNAs-comprisessintentit,VORGLBMP3FFPP-human reference DNA is beta-
`
`actin-anducRAs.
`
`29.
`
`(currently amended) The system of claim 28, wherein said covalently-attached
`
`
`
`
`
`oligonucleotides comprise thesequences-efSEOID-NOS-+1Fsequence of SEQ ID NO:5.
`
`Geneoscopy Exhibit 1029, Page 4
`
`Geneoscopy Exhibit 1029, Page 4
`
`

`

`PATENT
`Attorney Docket No.: EXCT-31920/US-3/CON
`
`REMARKS
`
`Claims 1-2, 5-6, 8, 13-17 and 24-29 are pending and under examination in the present
`
`application. In the Office Action mailed July 6, 2015, the Examiner made the following
`
`objections andrejections:
`
`Claims 1-2, 5, 8, 13-14, 16-17 and 24-29 stand rejected under 35 U.S.C. § 103(a) as
`
`being unpatentable over BAKER (US 2003/0173284, published 9/18/2003), in view of,
`
`SHUBER(US 2002/0164631, published 11/7/2002) and FOTEDARetal. (Laboratory
`
`Diagnostic Techniques for Entamoeba Species, Clinical Microbiology Reviews, July 2007, p.
`
`511-532; as evidenced by MORGANet al. (Comparison of PCR and Microscopy for Detection
`
`of Cryptosporidium parvum in Human Fecal Specimens: Clinical Trial, J. Clin Micro, Apr. 1998,
`
`p. 995-998), in further view of VAN ENGELAND (WO2008084219, published 7/17/2008),
`
`ZOU,et al. (Highly Methylated Genes in Colorectal Neoplasia: Implications for Screening,
`
`Cancer Epidemiol Biomarkers Prevo 2007 Dec;16(12):2686-96.), NCBI ACCESSION NOs.
`
`X56134 (10/7/2008), AC009118 (3/18/2003), NM_006528 (5/2/2010), NG_007992 (5/7/2010),
`
`NM_001201 (3/5/2010) and NM_033360 (4/25/2010).
`
`Claim 6 is rejected under 35 U.S.C. § 103(a) as being unpatentable over BAKER,in view
`
`of SHUBER and FOTEDAR(as evidenced by MORGAN),in further view of VAN
`
`ENGELAND, ZOU, NCBI ACCESSION NOs. X56134 (10/7/2008), AC009118 (3/18/2003),
`
`NM_006528 (5/2/2010), NG_007992 (Snt2010), NM_001201 (3/5/2010) and NM_033360
`
`(4/25/2010), in further view of Qiagen (QIAamp DNAStool Mini Kit Handbook, 8/2001).
`
`Claim 15 is rejected under 35 U.S.C. § 103(a) as being unpatentable over BAKER,in
`
`view of SHUBER and FOTEDAR(as evidenced by MORGAN), in further view of VAN
`
`ENGELAND, ZOU, NCBI ACCESSION NOs. X56134 (10/7/2008), AC009118 (3/18/2003),
`
`NM_006528 (5/2/2010), NG_007992 (Snt2010), NM_001201 (3/5/2010) and NM_033360
`
`(4/25/2010), in further view of STEPHAN (US 2010/0293 130, published 11/18/2010,
`
`effective filing date 11/30/2006).
`
`Applicants note with appreciation that the Examiner has suggested amendmentto include
`
`all of the genes shownin the multi-marker stool test of Example 7. However, for the reasons
`
`discussed below, for business reasons and to advance prosecution, and reserving the right to
`
`prosecute the original or similar claims in one or more future applications, Applicants herein
`
`Geneoscopy Exhibit 1029, Page 5
`
`Geneoscopy Exhibit 1029, Page 5
`
`

`

`PATENT
`Attorney Docket No.: EXCT-31920/US-3/CON
`
`amend Claim 1 to recite “... a plurality of different magnetic target-specific capture particles,
`
`wherein different target-specific capture particles comprise covalently-attached oligonucleotides
`
`complementary to at least a portion of different target human DNAs, and_wherein said different
`
`target human DNAs comprise VDRG4, BMP3, and KRAS”’.
`
`The claimsare not obvious
`
`The Examinerasserts that all of vimentin, NDRG4, BMP3, TFPI2, and KRAS were
`
`known colorectal cancer markers, and beta-actin was a familiar control gene at the time of the
`
`invention, and that a skilled artisan at the time of the invention would havehad aninterest in all
`
`of the above genesin order to detect colorectal cancer.
`
`Applicants respectfully submit that the cited art does not teach or suggest selection of the
`
`particular combination of ‘genes ofinterest’ recited in the claims as amended,northe particular
`
`combination of reagents used to assemble a sufficiently successful stool DNA assay for the
`
`detection of colorectal cancer. In contrast, Applicants have now shownthatthe particular
`
`combination of target human DNAsand system components for capturing them provide an the
`
`basis for an assay that has received regulatory approval. Specifically, the system components of
`
`instant claim 1 as amendedare included in Applicants’ product, the “Cologuard®” colorectal
`
`cancer screening test, whichis the first stool DNA screeningtest for colorectal cancer to receive
`
`FDAapproval. Thecited art does not teach or suggest the particular combination of NDRG4,
`
`BMP3 and KRAStarget human DNAs,or the collected set of system components to perform the
`
`steps to isolate these particular target human DNAsfrom humanstool samples.
`
`Provided herewith are the following documents:
`
`1.
`
`FDAPress Release “FDA approvesfirst non-invasive DNA screening test for colorectal
`
`cancer.”” August 8, 2014; announcing approval of the “Cologuard®” stool DNA test
`
`product;
`
`2.
`
`A set of instructions for the commercial assay entitled “Cologuard® sDNA-based
`
`Colorectal Cancer Screening Test Instructions for Use” (“Instructions’’)
`
`(a draft version
`
`of whichis also available for download on the World Wide Webat
`
`fda.gov/downloads/AdvisoryCommittees/CommitteesMeetingMaterials/MedicalDevices/
`
`Geneoscopy Exhibit 1029, Page 6
`
`Geneoscopy Exhibit 1029, Page 6
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`

`

`PATENT
`Attorney Docket No.: EXCT-31920/US-3/CON
`
`MedicalDevicesAdvisoryCommittee/MolecularandClinicalGeneticsPanel/UCM390231.p
`
`df), providing a complete description of the FDA-approved screeningtest.
`
`Asstated in the second paragraph ofthe “Principles of the Procedure” beginning
`
`on page8, the target human DNAs are NDRG4, BMP3, KRAS, anda reference gene
`
`(beta actin, ACTB):
`
`
`
`(Instructions for Use, page 8)
`
`This document further shows that the FDA-approved test comprises the system
`
`componentsrecited in the instant claims as amended: stool homogenization buffer, PVPP
`
`inhibitor adsorbant, spin filter and guanidine thiocyanate (see Instructions, page 11, items
`
`200204, 200151, 200138, and 200121, respectively), and magnetic target-specific capture
`
`particles for capturing NDRG4, BMP3 and KRAStarget human DNAs(“Capture
`
`Beads’’, Instructions, page 9, item 200150).
`
`ThomasF. Imperiale, M.D., et al., “Multitarget Stool DNA Testing for Colorectal-Cancer
`
`Screening”, New England Journal of Medicine 370(14):1287 (2014).
`
`This documentreports on the clinicaltrial of the Cologuard® test. Page 6 of the
`
`Appendix, “Supplemental Figure Legend” for Figure S1 showsthat the method used in
`
`the study comprised use of system components accordingto instant claim 1 as amended:
`
`Geneoscopy Exhibit 1029, Page 7
`
`Geneoscopy Exhibit 1029, Page 7
`
`

`

`PATENT
`Attorney Docket No.: EXCT-31920/US-3/CON
`
`
`
`poliapyrroticone io remove POR inisbiiors and sain fitered to clarty. Seuuence spectis DRA Siomarker targeis were istated airectly fram the
`
`eared supsrnigiant using 3 magneic bead based ciiggauctectds hybrid capture method. The captured DON¢ was split and ons portion was
`
`
`
`subjected f the insulfite soacthon for ihe idestitieation of aiseranty methylaied VoRts and BWacd ihe otter portion was used to determine tre
`
`
`presence of any af saven Hons print mutations. The tes! does not distinguish the specific mutation. Using quantifative reattime taeget and simai
`
`(Imperiale, et al., Supplementary Appendix, page 6)
`
`The data published by Imperiale showthat:
`
`e
`
`e
`
`e
`
`Sensitivity of the Cologuard assay in detecting patients with colorectal cancer was
`
`92% versus 74% for FIT (fecal immunochemicaltest) ;
`
`Sensitivity in detecting patients with colorectal cancers in Stages I-III] —those
`
`determined by the American Joint Committee on Cancerto be associated with an
`
`increased rate of being cured—was 93% for Cologuard versus 73 % for FIT;
`
`Sensitivity for patients with advanced pre-cancerouslesions was 42% for Cologuard
`
`versus 24% for FIT;
`
`e Cologuard detected 69% of patients with polyps with high-grade dysplasia versus
`
`46% for FIT;
`
`e Cologuard achieved a specificity of 87% versus FIT specificity of 95%.
`
`(Imperiale, page 1293, col 1 and Table 1).
`
`Long felt need andthefailure of others
`
`Asevidenced by the art cited by the Examiner,isolation of human DNAfrom stool
`
`samples for use in colorectal cancer screening has long been a goalin this field (see, e.g.,
`
`SHUBERascited above, published in 2002; ZOU and VAN ENGELAND, published in 2007
`
`and 2008, respectively), and different combinations of markers to detect colorectal cancer in
`
`stool samples have beentried (see, e.g., ZOU and VAN ENGELAND).
`
`VAN ENGELANDprovidesa detailed summary ofthe state of the art of isolating DNA
`
`from stool samples in 2008, and highlights that these methods are unsatisfactory:
`
`Geneoscopy Exhibit 1029, Page 8
`
`Geneoscopy Exhibit 1029, Page 8
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`

`

`PATENT
`Attorney Docket No.: EXCT-31920/US-3/CON
`
`
` fon hechniques typos
`4 a Sot ey
`~
`j
`ee x
`
`4g stool and more convenidentl
`¥ purified the
`
`2
`he
`une
`2
`a4
`.
`ew ene sd ae
`
`RTOS They qraebkic SHG GIRS
`Deng at al
`a
`wotroemeac
`Luprovement
`oe
`oo Can ye
`ss
`=a ES oe
`campriséed an slectrophoar
`
`
`sechientes, using oligomucteoctad
`ty = >
`mse)
`ts
`+
`tes.
`
`in an aorylamicd
`hat
`
`Lo Ds
`important to prevent degradation daring samole handling.
`
`improvec results were obtained with stool samples frozen as
`waco ley
`ome Meee at ae
`ms.
`-
`me] Deer a eee
`quickly a8 possibile after callection.
`5
`
`
`
`
`stabilis ko the stool ss
`Me
` 7
`An
`at
`Bre
`4 2Scenn
`further tranapork (Clann et al.,
`merit
`Foy RFE ‘Gq yoeS
`neni iiimrevel
`
`
`> OF an MSD celumn to
`
`Po pee
`ONE
`ye
`ESE
`On ee
`Se eg 7
`3
`Fr
`be
`“a
`7
`age
`pe
`ore
`AB
`a om doe
`current
`tosis For cancer cetectiorn in Faemed SEMELSS ARS
`
`
`(VAN ENGELAND, page6, line 21 to page 7, line 7, emphasis added)
`
`The references cited in making the rejection, including SHUBER, ZOU, and VAN
`
`ENGELAND, demonstrate that there has been a long-felt need for a way to screen for colorectal
`
`cancers based on stool DNAtesting. However, even in possession of the methods and markers
`
`disclosed in the art cited by the Examiner, as stated by VAN ENGELAND, other workersin the
`
`field have failed. In contrast, Applicants have been able to meet this need and succeed where
`
`others had failed by using a particular combination of markers and methods for extracting the
`
`marker DNA,as described in the instant application. The embodiments as claimed herein
`
`addressed this long-felt need and succeeded where others had failed, as evidenced by first-ever
`
`FDAapprovalfor the product containing these components.
`
`Accordingly, it is respectfully submitted that the claims are not obvious and the rejection
`
`is overcome, and Applicants respectfully request that the rejection be withdrawn.
`
`Geneoscopy Exhibit 1029, Page 9
`
`Geneoscopy Exhibit 1029, Page 9
`
`

`

`PATENT
`Attorney Docket No.: EXCT-31920/US-3/CON
`
`CONCLUSION
`
`For the reasonsset forth above,it is respectfully submitted that all objections and
`
`rejections have been addressed, and Applicants’ claims should be passed to allowance. Should
`
`the Examinerbelieve that a telephone interview would aid in the prosecution ofthis application,
`
`Applicants encourage the Examinerto call the undersigned collect at (608) 662-1277.
`
`Dated:
`
`July 31, 2015
`
`/Mary Ann D. Brow/
`Mary Ann D. Brow
`Registration No. 42,363
`
`CASIMIR JONES, S.C.
`2275 Deming Way,Suite 310
`Middleton, Wisconsin 53562
`608.662.1277
`
`-10-
`
`Geneoscopy Exhibit 1029, Page 10
`
`Geneoscopy Exhibit 1029, Page 10
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`

`

`
`
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`ESTABLISHED IN 1812
`
`APRIL 3, 2014
`
`VOL. 370 NO. 14
`
`Multitarget Stool DNA Testing for Colorectal-Cancer Screening
`
`
`ABSTRACT
`
`SACKGROUNDS
`
`An accurate, noninvasive test could improve the effectiveness of colorectal-cancer
`screening.
`
`METHOOS
`
`We compareda noninvasive, multitarget stool DNA test with a fecal immunochem-
`ical test (FIT) in personsat averagerisk for colorectal cancer. The DNAtest includes
`quantitative molecular assays for KRAS mutations, aberrant NDRG4 and BMP3 meth-
`ylation, and B-actin, plus a hemoglobin immunoassay. Results were generated with
`the use ofa logistic-regression algorithm, with values of 183 or more considered to
`be positive. FIT values of more than 100 ng of hemoglobin per milliliter of buffer
`were consideredto be positive. Tests were processed independently of colonoscopic
`findings.
`
`Of the 9989 participants who could be evaluated, 65 (0.7%) had colorectal cancer
`and 757 (7.6%) had advanced precancerous lesions (advanced adenomasorsessile
`serrated polyps measuring >1 cm in the greatest dimension) on colonoscopy. The
`sensitivity for detecting colorectal cancer was 92.3% with DNAtesting and 73.8%
`with FIT (P=0.002). The sensitivity for detecting advanced precancerouslesions was
`42.4% with DNA testing and 23.8% with FIT (P<0.001). The rate of detection of
`polyps with high-grade dysplasia was 69.2% with DNAtesting and 46.2% with FIT
`(P=0.004); the rates of detection of serrated sessile polyps measuring 1 cm or more
`were 42.4%and 5.1%, respectively (P<0.001). Specificities with DNA testing and FIT
`were 86.6% and 94.9%, respectively, among participants with nonadvancedor neg-
`ative findings (P<0.001) and 89.8% and 96.4%, respectively, among those with
`negative results on colonoscopy (P<0.001). The numbers of persons who would
`need to be screened to detect one cancer were 154 with colonoscopy, 166 with DNA
`testing, and 208 with FIT.
`
`CONCLUSIONS
`
`In asymptomatic persons at average risk for colorectal cancer, multitarget stool
`DNAtesting detected significantly more cancers than did FIT but had morefalse
`positive results. (Funded by Exact Sciences; ClinicalTrials.gov number, NCT01397747,)
`
`From the Department of Medicine,Indi-
`ana University School of Medicine, the
`Regenstrief Institute, the Simon Cancer
`Center, and the Center for Innovation at
`Roudebush Veterans Affairs Medical
`
`in Indianapolis (T.F.I.); the
`Center — all
`Departments of Medicine and Epidemi-
`ology and the Lineberger Comprehensive
`Cancer Center, University of North Caro-
`lina at Chapel Hill, Chapel Hill (D.F.R.); the
`Dr. Henry D. Janowitz Division of Gastro-
`enterology, Department of Medicine,
`Icahn School of Medicine at MountSinai,
`New York (S.H.1); Kaiser Permanente
`Medical Center, Walnut Creek, CA (T.R.L.);
`Boston Biostatistics Research Founda-
`
`tion, Framingham MA (P.L.); Exact Sci-
`ences, Madison, WI (G.P.L., B.M.B.); and
`the Division of Gastroenterology and
`Hepatology, Mayo Clinic, Rochester, MN
`(D.A.A.). Address reprint requests to Dr.
`Imperiale at Indiana University Medical
`Center-Regenstrief Institute, 1050 Wis-
`hard Blvd., Indianapolis, IN 46202.
`
`This article was published on March 19,
`2014, at NEJM.org.
`
`
`
`Copyright © 2014 Massachusetts Medical Society.
`
`N ENGL) MED 370;14 NEJM.ORG APRIL 3, 2014
`
`1287
`
`The New England Journal of Medicine
`Downloaded from nejm.org on July 15, 2015. For personal use only. No other uses wi Iu er
`Copyright © 2014 Massachusetts Medical Society. All rights reserved.
`
`$s10n.
`Exhibit 1029, Page 11
`
`Geneoscopy Exhibit 1029, Page 11
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`

`

`
`
`
`
`* OLORECTAL CANCER IS A MAJOR CAUSE
`of death and disease among men and
`women in the United States.1 The underly-
`ing neoplastic processes of colorectal carcino-
`genesis lend themselves to screening.? Evidence
`supports and guidelines endorse several tests and
`strategies,*> and screening for colorectal cancer
`has been found to be cost-effective.>7
`Despite the supporting evidence, recommenda-
`tions, and availability of several screeningtests,
`a substantial proportion of the U.S. population is
`not up to date with screening.® A simple, nonin-
`vasive test with high sensitivity for both colorectal
`cancer and advanced precancerouslesions might
`increase uptake and adherencerates, which could
`improve clinical outcomes.
`Colorectal cancer arises from accumulated
`genetic and epigenetic alterations, which provide
`a basis for the analysis of stool to identify tumor-
`specific changes.° Large-scale screening studies
`of previously available stool-based DNA tests
`showed only fair sensitivity for the detection of
`colorectal cancer (i.e., the capacity to detect can-
`cers, or true positive tests [see Glossary]) and low
`sensitivity for the detection of advanced adeno-
`mas,.1°11 Important advances have since been in-
`corporated, including the use of a stabilizing buf
`fer,1243 more discriminating markers,+1> more
`sensitive analytic methods,1+1%17 automation,"
`and an overall determination of results with the
`use of a logistic-regression algorithm, which to-
`getherresult in higher sensitivity for the detection
`of both cancer and advanced precancerous le-
`sions.1*16 However, evaluation of the more recent
`
`
`tests was based largely on analyses of archived
`specimens,
`including those collected from pa-
`tients after the diagnosis but before the resec-
`tion of colorectal cancer or advanced precancer-
`ous polyps.
`In this study, we evaluate the multitarget stool
`DNAtest as a tool for screening. The primary
`aim was to determine the performance charac-
`teristics of the DNAtest in the detection of colorec-
`tal cancer. The secondary aims were to deter-
`mine the performance of the DNA test in the
`detection of advanced precancerouslesions and
`to compare it with a commercially available fecal
`immunochemicaltest (FIT) for human hemoglo-
`bin in the detection of both colorectal cancer and
`advanced precancerouslesions.
`
`METHODS
`
`STUDY DESIGN
`
`From June 2011 through November2012, ween-
`rolled participants in this cross-sectional study at
`90 sites throughout the United States and Canada,
`including private-practice and academicsettings.
`The study was approved by the institutional review
`board at each site, and all participants provided
`written informed consent.
`The study, which was funded by Exact Sciences,
`was designed by the authors; Health Decisions,
`a contract research organization, gathered and
`monitored the data. The first author wrote the
`first draft of the manuscript, incorporating the
`other authors’ contributions; one of the authors,
`whois a statistician, analyzed the data and, along
`
`
`i
`Glossary of Screening Terms
`
`d: i
`
`tS
`
`Sensitivity (true positive rate): The proportion of persons with disease who have a positive test (positive test results among persons with
`disease).
`
`‘ Specificity (true negative rate): The proportion of persons without disease who havea negative test (negative test results among persons
`:
`without disease).
`
`: False negative rate (1 minus sensitivity): The proportion of persons with disease who have a negative test (negative test results among per-
`sons with disease).
`
`‘ False positive rate (1 minus specificity): The proportion of persons without disease who have a positive test (positive test results among
`persons without disease).
`
`‘ Positive predictive value: The proportion of persons with disease among those with a positive test (disease present among thosewith posi-
`:
`tive test results).
`
`‘ Negative predictive value: The proportion of persons without disease among those with a negative test (disease absent among those with
`:
`negative test results).
`$SaEEEEEE
`‘ Number needed to screen: The number of persons who would need to be screened to identify one person with the disease.
`
`1288
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`The New England Journal of Medicine
`G neosco
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`Copyright © 2014 Massachusetts Medical Society. All rights reserved.
`
`Geneoscopy Exhibit 1029, Page 12
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`

`

`MULTITARGET STOOL DNA TESTING FOR COLORECTAL CANCER
`
`with the last author, vouches for the data and
`adherence to the study protocol, which is avail-
`able with the full text of this article at NEJM.org.
`All the authors signed confidentiality agreements
`with Exact Sciences.
`
`STUDY POPULATION
`
`The target population was asymptomatic persons
`between the ages of 50 and 84 years who were
`considered to be at average risk for colorectal
`cancer and who were scheduled to undergo screen-
`ing colonoscopy. Enrollment was weighted toward
`persons 65 years of age or older in order to in-
`crease the prevalence of cancer. We excludedpar-
`ticipants who had a personalhistory of colorectal
`neoplasia, digestive cancer, or inflammatory bowel
`disease; had undergone colonoscopy within the
`previous 9 years or a barium enema, computed
`tomographic colonography, or sigmoidoscopy
`within the previous 5 years; had positive results
`on fecal blood testing within the previous 6 months;
`had undergonecolorectal resection for any reason
`other than sigmoid diverticula; had overt rectal
`bleeding within the previous 30 days; had a per-
`sonal or family history of colorectal cancer; had
`participated in any interventional clinical study
`within the previous 30 days; or were unable or
`unwilling to provide written informed consent.
`
`CLINICAL PROCEDURES
`
`All participants were required to provide a stool
`specimen and undergo screening colonoscopy
`within 90 days after providing informed consent.
`Stool was collected before routine bowel prepara-
`tion. No dietary or medication restrictions were
`required. Colonoscopists were required to describe
`the extent of the examination, document cecal
`visualization, rate the quality of preparation (on a
`modified Aronchick scale),1* and record the size
`and location oflesions.
`Although colonoscopists reported the location
`and size of all lesions, only the most advanced
`colorectal epithelial lesion (the index lesion) and
`its location (proximal or distal) were used to cat-
`egorize participants for the analysis. If two simi-
`larly advanced lesions were present, the larger of
`the two was designated as the index lesion. The
`proximal colon was considered to include the
`splenic flexure and all segments proximalto it,
`an insertion depth of more than 60 cm,or any
`
`part described by the phrase “right colon”; the
`distal colon was considered to include all other
`segments, an insertion depth of 60 cm orless,
`or any part described by the phrase “left colon.”
`The biopsy and surgical specimens underwent
`histopathological analysis at the laboratory typi-
`cally used by each study site. Polyps with high-
`grade dysplasia or 25%or more villous elements
`in adenomas measuring less than 1 cm, as well
`as sessile serrated or hyperplastic polyps measur-
`ing 1 cm orlarger, were re-reviewed centrally by
`a gastrointestinal pathologist for confirmation,
`with diagnostic disagreements resolved by con-
`sensus of at least two central pathologists.
`
`PRIMARY AND SECONDARY OUTCOMES
`
`The primary outcomewastheability of the DNA
`test to detect colorectal cancer (i.e., adenocarci-
`noma), with disease stage determined with the
`use of the American Joint Committee on Cancer
`(AJCC) staging system.The secondary outcome
`was the performance of the DNAtest for the detec-
`tion of advanced precancerouslesions, including
`advanced adenomas(high-grade dysplasia or with
`225% villous histologic features or measuring
`21 cm in the greatest dimension) andsessile ser-
`rated polyps measuring 1 cm or more in diameter.
`
`LABORATORY PROCEDURES
`
`A central biorepository receivedall stool specimens.
`Laboratory testing was performed without knowl-
`edge of the results of either the comparator FIT
`or clinical findings. (Details of stool collection
`and processing for DNAtesting are showninFig.
`S1 in the Supplementary Appendix, available at
`NEJM.org.) Buffered stool samples were homog-
`enized, separated into aliquots, and frozen at —80°C
`on receipt. Stool aliquots were subsequently sent
`in batches to one of three laboratories: Exact Sci-
`ences (Madison, WI), Mayo Medical Laboratory
`(Rochester, MN), and Molecular Pathology Labo-
`ratory Network (Knoxville, TN). Each laboratory
`received, in a blinded fashion, a similar distribu-
`tion of specimens on the basis of colonoscopic
`findings.
`The multitarget stool DNA test consists of
`molecular assays for aberrantly methylated BMP3
`and NDRG4 promoter regions, mutant KRAS, and
`B-actin (a reference gene for human DNA quan-
`tity), as well as an immunochemical assay for
`
`N ENGLJ MED 370;14
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`NEJM.ORG APRIL 3, 2014
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`The New England Journal of Medicine
`Downloaded from nejm.org on July 15, 2015. For personal use only. No other uses wi Outper
`Exnibit 1029, Page 13
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`Copyright © 2014 Massachusetts Medical Society. All rights reserved.
`
`Geneoscopy Exhibit 1029, Page 13
`
`

`

`human hemoglobin. Quantitative measurements
`of each marker were incorporated into a validat-
`ed, prespecified logistic-regression algorithm,
`with a value of 183 or more indicating that the
`test result was positive (for details, see the Sup-
`plementary Appendix). Analytic results were
`transferred to the study’s biostatistician.
`FIT (OC FIT-CHEK,Polymedco) was performed
`according to the manufacturer’s instructions with
`the use of the same stool sample used for the
`DNAtest.?° Samples were refrigerated on receipt
`and sent in batches to a separate single labora-
`tory for blinded analysis. Stool samples with
`more than 100 ng of hemoglobin per milliliter
`of buffer were considered to be positive.?°
`
`STATISTICAL ANALYSIS
`
`The study was designed to have a power of 90%
`to test the prespecified hypothesis that the DNA
`
`
`
`test would have a sensitivity of 65% or more for
`the detection of colorectal cancer (AJCC stages I
`through IV) underthe null hypothesis, at a one-
`sided typeI error rate of0.05. A secondary hypoth-
`esis was to rule out a 5%noninferiority margin
`for sensitivity for the detection of colorectal can-
`cer with the DNAtest as compared with FIT,at a
`one-sided type I error rate of 0.05. Testing of the
`two hypotheses with a powerof at least 80%re-
`quired the diagnosis of 49 and 56 adjudicated
`colorectal cancers, respectively, which required
`the enrollment of 10,500 to 12,000 participants,
`under the assumption of a colorectal-cancer preva-
`lence of4.5 cases per 1000 population.
`We conducted prespecified analyses to deter-
`mine the sensitivity of the multitarget DNAtest,
`as compared with FIT, for the detection of screen-
`ing-relevant colorectal cancer
`(AJCC stages I
`through III);
`the specificity of the multitarget
`A
`
`
`
`POLLLLLOLLILDDEDISLISLISLISEDESLISLISLISEDSDLISLISLISEDSEDISDISLISEISEDSEDISLISEISLDSEDISLISLISLDSEDESDISLISEDSEDDEDISLISEISLDSEDESDISLISLDDEDIEDISLISEDSEDEDISDISLISEDEEDISDISDISLDDEDESDISDISEDSEDEEDIDDISE:
`
`12,776 Participants provided written
`informed consent
`
`
`
`11,016 Could be evaluated
`
`1760 Could not be evaluated
`464 Withdrew consent
`1168 Did not undergo colonoscopy
`128 Did not submit stool sample
`
`
`
`
`
`
`34 Had FIT excluded because of
`
`689 Had multitarget DNAtest
`excluded
`474 Had stool samples that could
`not be evaluated owing to
`leakage in shipping or repeat
`specimen not received before
`colonoscopy
`213 Had technical failure owing
`to insufficient DNA (low
`B-actin), hemoglobin sample
`volume, stool supernatant for
`target capture, or material for
`repeat assay
`2 Had missing samples
`
`304 Had colonoscopy excluded
`194 Had negative but incomplete
`examinations
`94 Did not haveinsertion to
`cecum documented
`79 Had poor bowel preparation
`21 Had incomplete examination
`71 Underwent biopsy, but did not
`have pathology result owing to
`no tissue or loss of specimen
`20 Underwent colonoscopy before
`stool collection
`19 Underwent colonoscopy >90 days
`after enrollment
`
`9989 Wereincluded in the primary
`analysis
`65 Had colorectal cancer
`757 Had advanced precancerous
`lesions
`2893 Had nonadvanced adenoma
`6274 Had negative results
`
`insufficient hemoglobin sample
`
`ot
`S
`
`ve -¥. Enrollment and Outcomes.
`
`N ENGL) MED 370314
`
`NEJM.ORG
`
`APRIL 3, 2014
`
`The New England Journal of Medicine
`Genegscopy
`Downloaded from nejm.org o