Multitarget Stool DNA Testing for Colorectal-Cancer Screening
`Thomas F. Imperiale, M.D., David F. Ransohoff, M.D., Steven H. Itzkowitz, M.D., Theodore R. Levin, M.D.,
`Philip Lavin, Ph.D., Graham P. Lidgard, Ph.D., David A. Ahlquist, M.D., and Barry M. Berger, M.D.
`
`A BS TR AC T
`
`Background
`An accurate, noninvasive test could improve the effectiveness of colorectal-cancer
`screening.
`
`Methods
`We compared a noninvasive, multitarget stool DNA test with a fecal immunochem-
`ical test (FIT) in persons at average risk for colorectal cancer. The DNA test includes
`quantitative molecular assays for KRAS mutations, aberrant NDRG4 and BMP3 meth-
`ylation, and β-actin, plus a hemoglobin immunoassay. Results were generated with
`the use of a 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 considered to be positive. Tests were processed independently of colonoscopic
`findings.
`
`Results
`Of the 9989 participants who could be evaluated, 65 (0.7%) had colorectal cancer
`and 757 (7.6%) had advanced precancerous lesions (advanced adenomas or sessile
`serrated polyps measuring ≥1 cm in the greatest dimension) on colonoscopy. The
`sensitivity for detecting colorectal cancer was 92.3% with DNA testing and 73.8%
`with FIT (P = 0.002). The sensitivity for detecting advanced precancerous lesions 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 DNA testing 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 nonadvanced or 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
`DNA testing detected significantly more cancers than did FIT but had more false
`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
`Center — all in Indianapolis (T.F.I.); the
`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 Mount Sinai,
`New York (S.H.I.); 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.
`
`N Engl J Med 2014;370:1287-97.
`DOI: 10.1056/NEJMoa1311194
`Copyright © 2014 Massachusetts Medical Society.
`
`1287
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`n engl j med 370;14 nejm.org april 3, 2014
`
`The new england
`journal of medicine
`
`established in 1812
`
`april 3, 2014
`
`vol. 370 no. 14
`
`The New England Journal of Medicine
`
`Downloaded from nejm.org on December 5, 2023. For personal use only. No other uses without permission.
`
` Copyright © 2014 Massachusetts Medical Society. All rights reserved.
`
`Patent Owner's Ex. 2008, Page 1
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`Geneoscopy Exhibit 1077, Page 1
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`

`

`Colorectal 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.2 Evidence
`supports and guidelines endorse several tests and
`strategies,3-5 and screening for colorectal cancer
`has been found to be cost-effective.5-7
`Despite the supporting evidence, recommenda-
`tions, and availability of several screening tests,
`a substantial proportion of the U.S. population is
`not up to date with screening.8 A simple, nonin-
`vasive test with high sensitivity for both colorectal
`cancer and advanced precancerous lesions might
`increase uptake and adherence rates, 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.9 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.10,11 Important advances have since been in-
`corporated, including the use of a stabilizing buf-
`fer,12,13 more discriminating markers,14,15 more
`sensitive analytic methods,14,16,17 automation,16
`and an overall determination of results with the
`use of a logistic-regression algorithm, which to-
`gether result in higher sensitivity for the detection
`of both cancer and advanced precancerous le-
`sions.14,16 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
`DNA test as a tool for screening. The primary
`aim was to determine the performance charac-
`teristics of the DNA test 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 precancerous lesions and
`to compare it with a commercially available fecal
`immunochemical test (FIT) for human hemoglo-
`bin in the detection of both colorectal cancer and
`advanced precancerous lesions.
`
`Me thods
`
`Study Design
`From June 2011 through November 2012, we en-
`rolled participants in this cross-sectional study at
`90 sites throughout the United States and Canada,
`including private-practice and academic settings.
`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,
`who is a statistician, analyzed the data and, along
`
`Glossary of Screening Terms
`
`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 have a 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 those with 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).
`
`Number needed to screen: The number of persons who would need to be screened to identify one person with the disease.
`
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`n engl j med 370;14 nejm.org april 3, 2014
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`The New England Journal of Medicine
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`Downloaded from nejm.org on December 5, 2023. For personal use only. No other uses without permission.
`
` Copyright © 2014 Massachusetts Medical Society. All rights reserved.
`
`Patent Owner's Ex. 2008, Page 2
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`Geneoscopy Exhibit 1077, Page 2
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`

`

`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 excluded par-
`ticipants who had a personal history 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 undergone colorectal 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),18 and record the size
`and location of lesions.
`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 proximal to 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 or less,
`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 or larger, 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 outcome was the ability 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.19 The secondary outcome
`was the performance of the DNA test for the detec-
`tion of advanced precancerous lesions, including
`advanced adenomas (high-grade dysplasia or with
`≥25% villous histologic features or measuring
`≥1 cm in the greatest dimension) and sessile ser-
`rated polyps measuring 1 cm or more in diameter.
`
`Laboratory Procedures
`A central biorepository received all 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 DNA testing are shown in Fig.
`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
`β-actin (a reference gene for human DNA quan-
`tity), as well as an immunochemical assay for
`
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`The New England Journal of Medicine
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`Downloaded from nejm.org on December 5, 2023. For personal use only. No other uses without permission.
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`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
`DNA test.20 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.20
`
`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) under the null hypothesis, at a one-
`sided type I error rate of 0.05. A secondary hypoth-
`esis was to rule out a 5% noninferiority margin
`for sensitivity for the detection of colorectal can-
`cer with the DNA test as compared with FIT, at a
`one-sided type I error rate of 0.05. Testing of the
`two hypotheses with a power of 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 of 4.5 cases per 1000 population.
`We conducted prespecified analyses to deter-
`mine the sensitivity of the multitarget DNA test,
`as compared with FIT, for the detection of screen-
`ing-relevant colorectal cancer (AJCC stages I
`through III); the specificity of the multitarget
`
`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
`insufficient hemoglobin sample
`
`689 Had multitarget DNA test
` 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
`β-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 have insertion 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 Were included in the primary
`analysis
`65 Had colorectal cancer
`757 Had advanced precancerous
`lesions
`2893 Had nonadvanced adenoma
`6274 Had negative results
`
`Figure 1. Enrollment and Outcomes.
`
`1290
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`n engl j med 370;14 nejm.org april 3, 2014
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`The New England Journal of Medicine
`
`Downloaded from nejm.org on December 5, 2023. For personal use only. No other uses without permission.
`
` Copyright © 2014 Massachusetts Medical Society. All rights reserved.
`
`
`
`
`
`
`
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`

`

`DNA stool test (i.e., true negative rate), with ad-
`vanced precancerous lesions on colonoscopy ex-
`cluded and only nonadvanced adenomas and
`negative results included (the primary measure
`of specificity) and with only negative results in-
`cluded (the secondary measure of specificity);
`and the sensitivity of the multitarget stool DNA
`test, as compared with FIT, for the detection of
`advanced precancerous lesions. The analyses
`were based on data from all participants who had
`valid results on multitarget stool DNA testing,
`FIT, and colonoscopy; all reported subgroup
`analyses were prespecified.
`For test characteristics, 95% lower boundar-
`ies were computed with the use of an exact bi-
`nomial test. Lower 95% confidence limits for
`comparative analyses were computed with the
`use of a one-sided McNemar paired-comparisons
`test for the observed difference in sensitivity be-
`tween the DNA test and FIT. The Hanley–McNeil
`method was used to calculate P values for the
`analysis of the receiver operating characteristic
`(ROC) curve.21 There were no interim analyses
`of the data. All analyses were conducted with the
`use of SAS software, version 9.1, and StatXact
`software, version 7.
`
`R esults
`
`Study Population
`A total of 12,776 participants were enrolled at 90
`sites; 9989 of these participants (78.2%) had re-
`sults that could be fully evaluated (Fig. 1). The par-
`ticipants whose results could be fully evaluated
`and those whose results could not be fully evalu-
`ated differed significantly with respect to mean
`age and race, although the magnitudes of the dif-
`ferences were small (Table S1 in the Supplemen-
`tary Appendix).
`A total of 65 participants who could be
`evaluated were found to have colorectal cancer
`on colonoscopy (prevalence, 0.7%). Of these par-
`ticipants, 60 had screening-relevant (stage I to
`III) cancers. A total of 757 participants who
`could be evaluated had advanced precancerous
`lesions (prevalence, 7.6%).
`
`DNA Test Characteristics
`Multitarget stool DNA testing identified 60 of 65
`participants with cancer, including 56 of the 60
`participants with screening-relevant cancers, for
`respective sensitivities of 92.3% (95% confidence
`interval [CI], 83.0 to 97.5) and 93.3% (95% CI,
`
`Table 1. Sensitivity and Specificity of the Multitarget Stool DNA Test and the Fecal Immunochemical Test (FIT)
`for the Most Advanced Findings on Colonoscopy.
`
`Most Advanced Finding
`
`Colonoscopy
`(N = 9989)
`
`Multitarget DNA Test
`(N = 9989)
`
`FIT
`(N = 9989)
`
`no.
`
`65
`
`60
`
`104
`
`757
`
`2893
`
`Positive
`Results
`
`no.
`
`Sensitivity
`(95% CI)
`
`%
`
`Positive
`Results
`
`no.
`
`Sensitivity
`(95% CI)
`
`%
`
`60
`
`56
`
`87
`
`321
`
`498
`
`92.3 (83.0–97.5)
`
`93.3 (83.8–98.2)
`
`83.7 (75.1–90.2)
`
`42.4 (38.9–46.0)
`
`17.2 (15.9–18.6)
`
`Specificity
`(95% CI)
`
`48
`
`44
`
`66
`
`180
`
`220
`
`73.8 (61.5–84.0)
`
`73.3 (60.3–83.9)
`
`63.5 (53.5–72.7)
`
`23.8 (20.8–27.0)
`
`7.6 (6.7–8.6)
`
`Specificity
`(95% CI)
`
`9167
`
`1231
`
`86.6 (85.9–87.2)
`
`472
`
`94.9 (94.4–95.3)
`
`Colorectal cancer
`
`Any
`
`Stage I to III*
`
`Colorectal cancer and
`high-grade dysplasia
`
`Advanced precancerous lesions†
`
`Nonadvanced adenoma
`
`All nonadvanced adenomas,
`non-neoplastic findings,
`and negative results on
`colonoscopy
`
`Negative results on colonoscopy
`
`4457
`
`455
`
`89.8 (88.9–90.7)
`
`162
`
`96.4 (95.8–96.9)
`
`* These stages of colorectal cancer, as defined by the system recommended by the American Joint Committee on Cancer,
`are associated with an increased rate of cure.
`† Advanced precancerous lesions include advanced adenomas and sessile serrated polyps measuring 1 cm or more.
`
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`n engl j med 370;14 nejm.org april 3, 2014
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`The New England Journal of Medicine
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`Downloaded from nejm.org on December 5, 2023. For personal use only. No other uses without permission.
`
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`
`Patent Owner's Ex. 2008, Page 5
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`Geneoscopy Exhibit 1077, Page 5
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`

`

`Multitarget DNA test
`
`FIT
`
`P=0.002
`
`P=0.04
`
`P=0.06
`
`P<0.001
`
`P<0.001
`
`Sensitivity(%)
`
`Proxim alCancer
`
`A ColorectalCancerAccordingtoStage
`P=0.06
`
`P=0.04
`
`100
`90
`80
`70
`60
`50
`40
`30
`20
`10
`0
`
`Sensitivity(%)
`
`Stage
`I
`(N=29)
`
`Stage
`II
`(N=21)
`
`Stage
`III
`(N=10)
`
`Stage
`IV
`(N=4)
`
`Stage
`I–III
`(N=60)
`
`B CancerandAdvancedPrecancerousLesionsAccordingto
`Location
`100
`90
`80
`70
`60
`50
`40
`30
`20
`10
`0
`
`DistalAdvanced
`Proxim alAdvanced
`
`PrecancerousLesionsPrecancerousLesions
`DistalCancer
`(N =30)
`(N =35)
`
`(N =431)
`
`(N =325)
`
`C Higher-RiskTypesamongAdvancedPrecancerousLesions D AdvancedPrecancerousLesionsAccordingtoSize
`ofLargestLesion
`100
`90
`80
`70
`60
`50
`40
`30
`20
`10
`
`P value for trend:
`Multitarget DNA Test, P<0.001
`FIT, P<0.001
`
`Sensitivity(%)
`
`≥3.0cm
`(N =38)
`
`
`(N =10)
`
`0 ≤
`
`
`
`
`2.0to2.9cm>0.5to<1.0cm0.5cm 1.0to1.9cm
`(N =79)
`(N =56)
`(N =574)
`
`High-Grade
`Dysplasia
`(N=39)
`
`SessileSerrated
`Polyp≥1.0cm
`(N=99)
`
`100
`90
`80
`70
`60
`50
`40
`30
`20
`10
`0
`
`Sensitivity(%)
`
`Figure 2. Sensitivity of the Multitarget Stool DNA Test and the Commercial Fecal Immunochemical Test (FIT),
`According to Subgroup.
`Shown are the sensitivities of the DNA test and FIT for the detection of colorectal cancer according to tumor stage
`(Panel A), for the detection of colorectal cancer and advanced precancerous lesions according to the location in the
`colon (Panel B), and for the detection of higher-risk subtypes among participants with advanced precancerous le-
`sions (Panel C) and according to lesion size (Panel D). The numbers in parentheses are the number of participants
`in each category. In Panel A, the stage of 1 of 65 colorectal cancers was not available. In Panel B, the location of
`1 of 757 advanced precancerous lesions was not available.
`
`83.8 to 98.2) (Table 1). Sensitivity did not vary
`significantly according to cancer stage (Fig. 2A)
`or location within the colon (Fig. 2B). Among
`757 participants with advanced precancerous le-
`sions, DNA testing detected 321 (42.4%; 95% CI,
`38.9 to 46.0). A total of 69.2% (95% CI, 52.4 to
`83.0) of 39 participants with high-grade dyspla-
`sia and 42.4% (95% CI, 32.6 to 52.8) of 99 par-
`ticipants with sessile serrated polyps measuring
`
`1 cm or larger were identified on DNA testing
`(Fig. 2C). The sensitivity of the DNA test was
`higher for distal advanced precancerous lesions
`(177 of 325 [54.5%; 95% CI, 48.9 to 60.0]) than
`for proximal lesions (143 of 431 [33.2%; 95% CI,
`28.8 to 37.8]) (Fig. 2B); test sensitivity increased
`as the lesion size increased (Fig. 2D). The sensi-
`tivity for the detection of cancer or advanced pre-
`cancerous lesions did not differ significantly ac-
`
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`n engl j med 370;14 nejm.org april 3, 2014
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`The New England Journal of Medicine
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`Downloaded from nejm.org on December 5, 2023. For personal use only. No other uses without permission.
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` Copyright © 2014 Massachusetts Medical Society. All rights reserved.
`
`Patent Owner's Ex. 2008, Page 6
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`

`

`1293
`
`AreaunderROCCurve
`Multitarget DNA test (0.94)
`FIT (0.89)
`
`A ColorectalCancer
`1.00
`
`0.75
`
`0.50
`
`0.25
`
`Sensitivity
`
`0.00
`0.00
`
`0.25
`
`0.50
`1−Specificity
`
`0.75
`
`1.00
`
`B AdvancedColorectalNeoplasia
`1.00
`
`AreaunderROCCurve
`Multitarget DNA test (0.73)
`FIT (0.67)
`
`0.75
`
`0.50
`
`0.25
`
`Sensitivity
`
`0.00
`0.00
`
`0.25
`
`0.50
`1−Specificity
`
`0.75
`
`1.00
`
`Figure 3. Receiver Operating Characteristic (ROC)
`Curves Comparing DNA Testing and FIT for the
`Detection of Colorectal Cancer and Advanced
`Colorectal Neoplasia.
`Shown are ROC curves for the multitarget stool DNA
`test and FIT for the detection of colorectal cancer
`(Panel A) and advanced colorectal neoplasia (colo-
`rectal cancer plus advanced precancerous lesions)
`(Panel B). For colorectal cancer, the area under the
`ROC curve was 0.94 for the DNA test and 0.89 for
`FIT (95% confidence interval [CI] for the difference in
`area, 0.003 to 0.10; P = 0.04). For advanced colorectal
`neoplasia, the area under the ROC curve was 0.73 for
`the DNA test and 0.67 for FIT (95% CI for the differ-
`ence in area, 0.04 to 0.09; P<0.001). The respective
`performance thresholds were a value of 183 or more
`for the DNA test and more than 100 ng of hemoglobin
`per milliliter of buffer for FIT.
`
`cording to age or laboratory-testing site (data not
`shown).
`Among 9167 participants who had findings
`other than colorectal cancer or advanced precan-
`cerous lesions (e.g., nonadvanced adenomas or
`negative results), the specificity of the DNA test
`(true negative rate) was 86.6% (95% CI, 85.9 to
`87.2). Among the 4457 participants with totally
`negative results on colonoscopy, the specificity
`was 89.8% (95% CI, 88.9 to 90.7); within this
`subgroup, the specificity was 94.0% among par-
`ticipants younger than 65 years of age and 87.1%
`among those 65 years of age or older (P<0.001).
`
`Comparison with FIT
`FIT detected 48 of 65 cancers (73.8%; 95% CI,
`61.5 to 84.0), 44 of 60 AJCC stage I to III cancers
`(73.3%; 95% CI, 60.3 to 83.9), and 180 of 757
`advanced precancerous lesions (23.8%; 95% CI,
`20.8 to 27.0), findings that were all significantly
`inferior to those with DNA testing (Table 1). FIT
`detected 20 of 30 proximal cancers (66.7%) and
`28 of 35 distal cancers (80.0%) (P = 0.35 for the
`comparison between proximal and distal location).
`Comparative results for the detection of cancer
`according to stage and for higher-risk subsets of
`advanced precancerous lesions are shown in Fig-
`ure 2. The DNA test was more sensitive than FIT
`for the detection of lesions with high-grade dys-
`plasia (69.2% vs. 46.2%, P = 0.004) or sessile ser-
`rated polyps measuring 1 cm or more (42.4% vs.
`5.1%, P<0.001) (Fig. 2C) and for the detection of
`advanced precancerous lesions within the size
`ranges observed (Fig. 2D).
`DNA testing detected 13 of 60 screening-rel-
`evant cancers that were undetected by FIT, where-
`as FIT detected 1 cancer that was undetected by
`DNA testing (P<0.001). DNA testing detected 170
`of 757 advanced precancerous lesions (22.5%) that
`were undetected by FIT, whereas FIT detected 29
`such lesions (3.8%) undetected by DNA testing
`(P<0.001).
`Among 9167 participants with findings
` other than colorectal cancer or advanced pre-
`cancerous lesions, the specificity of FIT was
`94.9% (95% CI, 94.4 to 95.3). Among 4457 par-
`ticipants with negative results on colonoscopy,
`the specificity was 96.4% (95% CI, 95.8 to 96.9).
`In these two subgroups, the specificity values
`were superior to those of the DNA test (Table 1).
`
`Multitarget Stool DNA Testing for Colorectal Cancer
`
`n engl j med 370;14 nejm.org april 3, 2014
`
`The New England Journal of Medicine
`
`Downloaded from nejm.org on December 5, 2023. For personal use only. No other uses without permission.
`
` Copyright © 2014 Massachusetts Medical Society. All rights reserved.
`
`Patent Owner's Ex. 2008, Page 7
`
`Geneoscopy Exhibit 1077, Page 7
`
`

`

`The specificity of FIT varied minimally accord-
`ing to age.
`As measured by the area under the ROC curve
`(AUC), the discrimination between colorectal can-
`cer and the combination of nonadvanced neopla-
`sia and lesser findings was significantly higher
`with DNA testing than with FIT (0.94 vs. 0.89,
`P = 0.04) (Fig. 3A); the AUC values for discrimi-
`nation between advanced colorectal neoplasia
`(colorectal cancer plus advanced precancerous le-
`sions) and all other findings were 0.73 and 0.67,
`respectively (P<0.001) (Fig. 3B). Positive and nega-
`tive predictive values are shown in Table S2 in the
`Supplementary Appendix.
`The isolated performance of the hemoglobin
`immunoassay component of the multitarget DNA
`test was similar to that of FIT, with specificities
`of 94.8% and 94.9%, respectively; sensitivities
`were 72.3% and 73.8%, respectively, for the detec-
`tion of colorectal cancer and 22.7% and 23.8%,
`respectively, for the detection of advanced pre-
`cancerous lesions.
`Table 2 shows the number of persons who
`would need to be screened with colonoscopy,
`multitarget DNA testing, and FIT in order to
`detect one colorectal cancer (154 with colonos-
`copy, 166 with multitarget DNA testing, and 208
`with FIT) and to detect one advanced precancer-
`ous polyp (13, 31, and 55 persons, respectively).
`These calculations show that multitarget DNA
`testing detected clinically significant lesions
`more efficiently than FIT.
`
`Extrapolation to an Expanded Screening
`Population
`In an extrapolation of our results to a hypotheti-
`cal reference population of 10,000 participants at
`average risk for colorectal cancer, the various
`screening techniques of colonoscopy, DNA testing,
`and FIT would identify, respectively, 65, 60, and
`48 persons with colorectal cancer; 758, 321, and
`180 persons with advanced precancerous lesions;
`
`2896, 498, and 220 persons with nonadvanced
`adenomas; and 6281, 732, and 248 persons with
`non-neoplastic findings or negative results on colo-
`noscopy (Table 3).
`The protocol specified the detection of colorec-
`tal cancer and advanced precancerous polyps as
`positive findings and the detection of nonadvanced
`adenomas as negative findings. In the hypo-
`thetical reference population of 10,000 persons,
`the numbers of persons who would be referred
`for colonoscopy on the basis of positive test re-
`sults would be 1611 (16.1%) with DNA testing
`and 696 (7.0%) with FIT. Of the positive test re-
`sults, the numbers that would be viewed as false
`positives would be 1230 of 1611 (76.4%) with
`DNA testing and 468 of 696 (67.2%) with FIT. Of
`8389 negative results for DNA testing, 442 (5.3%)
`would be viewed as false negatives, consisting of
`5 cancers and 437 advanced precancerous pol-
`yps. Of 9304 negative results for FIT, 595 (6.4%)
`would be viewed as false negatives, consisting of
`17 cancers and 578 precancerous polyps. If non-
`advanced adenomas were considered to be posi-
`tive findings, then the proportions of positive
`tests viewed as false positives would be 732 of
`1611 (45.4%) with DNA testing and 248 of 696
`(35.6%) with FIT. The numbers of negative tests
`viewed as false negatives would be 2840 of 8389
`(33.9%) with DNA testing and 3271 of 9304
`(35.2%) with FIT. Most of these false negative
`results would be small, nonadvanced adenomas
`(in 2398 of 2840 participants [84.4%] with DNA
`testing and 2676 of 3271 participants [81.8%]
`with FIT), with only rare instances of colorectal
`cancers (5 of 2840 [0.2%] and 17 of 3271 [0.5%],
`respectively).
`
`Discussion
`
`We compared a multitarget stool DNA test with a
`commercial FIT among patients at average risk
`for colorectal cancer. The sensitivity of the DNA
`
`Table 2. Numbers of Persons Who Would Need to Be Screened with Colonoscopy, Multitarget DNA Test, and FIT
`to Detect One Colorectal Cancer and One Advanced Precancerous Lesion.
`
`Finding
`
`Any colorectal cancer
`
`Stage I to III colorectal cancer
`
`Advanced precancerous lesion
`
`Colonoscopy
`
`154 (120–200)
`
`166 (130–217)
`
`13 (12–14)
`
`Number Needed to Screen (95% CI)
`
`Multitarget DNA Test
`
`FIT
`
`166 (130–217)
`
`178 (140–238)
`
`31 (28–35)
`
`208 (156–286)
`
`227 (169–313)
`
`55 (48–65)
`
`1294
`
`T h e ne w e ngl a nd jou r na l o f m e dic i ne
`
`n engl j med 370;14 nejm.org april 3, 2014
`
`The New England Journal of Medicine
`
`Downloaded from nejm.org on December 5, 2023. For personal use only. No other uses without permission.
`
` Copyright © 2014 Massachusetts Medical Society. All rights reserved.
`
`Patent Owner's Ex. 2008, Page 8
`
`Geneoscopy Exhibit 1077, Page 8
`
`

`

`Table 3. Extrapolation of Findings to an Expanded Population of 10,000 Persons at Average Risk for Colorectal Cancer
`Undergoing Screening with Colonoscopy, Multitarget Stool DNA Test, and FIT.*
`
`Colonoscopy Finding
`
`Persons
`with Finding
`
`Colorectal cancer
`
`Advanced precancerous lesions
`
`Nonadvanced adenomas
`
`Negative results: no colorectal cancer,
`advanced precancerous lesions,
`or nonadvanced adenomas
`
`Multitarget DNA Test
`
`Positive
`Results
`(N = 1611)
`
`Negative
`Results
`(N = 8389)
`
`FIT
`
`Positive
`Results
`(N = 696)
`
`Negative
`Re