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`From the Department of Medicine, Indi-
`ana University, and the Regenstrief Insti-
`tute — both in Indianapolis, (T.F.I.); the De-
`partment of Medicine, University of North
`Carolina at Chapel Hill, Chapel Hill (D.F.R.);
`the Department of Medicine, Mount Sinai
`School of Medicine, New York (S.H.I.); Care-
`Stat, Newton, Mass. (B.A.T.); and Exact Sci-
`ences, Marlborough, Mass. (M.E.R.). Ad-
`dress reprint requests to Dr. Imperiale at the
`Regenstrief Institute, 1050 Wishard Blvd.,
`Indianapolis, IN 46202.
`
`*The members of the Colorectal Cancer
`Study Group are listed in the Appendix.
`
`N Engl J Med 2004;351:2704-14.
`Copyright © 2004 Massachusetts Medical Society.
`
` new england journal
`The
`
`of
`
` medicine
`
`original article
`
`Fecal DNA versus Fecal Occult Blood
`for Colorectal-Cancer Screening
`in an Average-Risk Population
`
`Thomas F. Imperiale, M.D., David F. Ransohoff, M.D., Steven H. Itzkowitz, M.D.,
`Barry A. Turnbull, Ph.D., and Michael E. Ross, M.D.,
`for the Colorectal Cancer Study Group*
`
`abstract
`
`background
`Although fecal occult-blood testing is the only available noninvasive screening method
`that reduces the risk of death from colorectal cancer, it has limited sensitivity. We com-
`pared an approach that identifies abnormal DNA in stool samples with the Hemoccult II
`fecal occult-blood test in average-risk, asymptomatic persons 50 years of age or older.
`
`methods
`Eligible subjects submitted one stool specimen for DNA analysis, underwent standard
`Hemoccult II testing, and then underwent colonoscopy. Of 5486 subjects enrolled, 4404
`completed all aspects of the study. A subgroup of 2507 subjects was analyzed, including
`all those with a diagnosis of invasive adenocarcinoma or advanced adenoma plus ran-
`domly chosen subjects with no polyps or minor polyps. The fecal DNA panel consisted
`of 21 mutations.
`
`results
`The fecal DNA panel detected 16 of 31 invasive cancers, whereas Hemoccult II iden-
`tified 4 of 31 (51.6 percent vs. 12.9 percent, P=0.003). The DNA panel detected 29 of
`71 invasive cancers plus adenomas with high-grade dysplasia, whereas Hemoccult II
`identified 10 of 71 (40.8 percent vs. 14.1 percent, P<0.001). Among 418 subjects with
`advanced neoplasia (defined as a tubular adenoma at least 1 cm in diameter, a polyp
`with a villous histologic appearance, a polyp with high-grade dysplasia, or cancer), the
`DNA panel was positive in 76 (18.2 percent), whereas Hemoccult II was positive in 45
`(10.8 percent). Specificity in subjects with negative findings on colonoscopy was 94.4
`percent for the fecal DNA panel and 95.2 percent for Hemoccult II.
`
`conclusions
`Although the majority of neoplastic lesions identified by colonoscopy were not detect-
`ed by either noninvasive test, the multitarget analysis of fecal DNA detected a greater
`proportion of important colorectal neoplasia than did Hemoccult II without compro-
`mising specificity.
`
`2704
`
`n engl j med
`
`351;26
`
`www.nejm.org december
`
`23, 2004
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`The New England Journal of Medicine
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`Downloaded from nejm.org on October 23, 2023. For personal use only. No other uses without permission.
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` Copyright © 2004 Massachusetts Medical Society. All rights reserved.
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`c
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`fecal dna for colorectal-cancer screening
`
`olorectal cancer is the second
`leading cause of death from cancer among
`1,2
`adults.
` Despite recommendations en-
`dorsing screening, less than 40 percent of people
`50 years of age or older undergo screening for colo-
`3
`rectal cancer.
` Guaiac-based chemical detection of
`fecal occult blood is the only noninvasive screening
`method with proven effectiveness, reducing both
`4
`the incidence
` and the risk of death from colorectal
`5-7
`cancer
` when used programmatically. However,
`the sensitivity of fecal occult-blood testing for colo-
`rectal cancer and especially for colorectal adeno-
`mas is low because neoplasms may not bleed and
`2
` The availabil-
`thus cannot be detected in this way.
`ity of a simple, noninvasive test that detects tumor-
`specific products with reasonable sensitivity and
`specificity might overcome barriers to screening
`among patients who are not willing to undergo
`more sensitive but more invasive tests, such as co-
`lonoscopy.
`The molecular genetics of colorectal cancer pro-
`8,9
`vides the basis for the analysis of fecal DNA.
`Eighty-five percent of colorectal cancers result from
`chromosomal instability, with mutations progres-
`sively accumulating in the adenomatous polyposis
`APC
`p53
`coli (
`) gene, the
` tumor-suppressor gene,
`10
`-ras
` oncogene.
` The other 15 percent arise
`and the K
`from a loss of genes involved in DNA-mismatch re-
`11
` Co-
`pair, manifested by microsatellite instability.
`lorectal cancer may also be detectable through the
`use of DNA markers associated with disordered
`12
`apoptosis.
`Previous studies using fecal-based DNA testing
`have reported a sensitivity of 62 to 91 percent for
`cancer and 27 to 82 percent for advanced adeno-
`mas, with a specificity of 93 to 96 percent in persons
`13-17
`with normal findings on colonoscopy.
` Howev-
`er, those studies assessed persons with advanced,
`symptomatic lesions. We made a head-to-head com-
`parison of a fecal-based, multitarget DNA panel
`with Hemoccult II in asymptomatic adults, 50 years
`of age or older, who were at average risk for colo-
`rectal cancer. The primary objective was to compare
`detection rates for colorectal cancer and for colorec-
`tal cancer plus adenomas with high-grade dysplasia.
`
`methods
`
`study design and rationale
`The rationale for the study was based on screen-
`ing guidelines indicating that newer screening tests
`need not demonstrate a reduction in cost-specific
`
`mortality but should be at least as sensitive, specific,
`and safe, among other features, as current screen-
`18
`ing tests.
` Hemoccult II (Beckman Coulter, for-
`merly SmithKline Diagnostics) was chosen for the
`comparison with the DNA panel because it is the
`only fecal occult-blood test proven to reduce the in-
`cidence and risk of death from colorectal cancer
`2
`and is the most widely used guaiac-based test.
` The
`study was designed by the authors, with advice from
`national experts on colorectal cancer, cancer screen-
`*
`ing and prevention, and study design.
`The study was conducted at 81 sites, including
`private-practice and university-based settings. Sub-
`jects were enrolled between August 2001 and March
`2003. All subjects first provided a fecal sample for
`DNA testing and then completed three Hemoccult II
`cards before undergoing screening colonoscopy.
`All tests were conducted in a blinded fashion. Stool
`samples were analyzed for DNA abnormalities with-
`out knowledge of Hemoccult II or colonoscopy re-
`sults; colonoscopy was performed without knowl-
`edge of the results of fecal DNA testing. Since
`Hemoccult II testing was conducted at the study
`sites, the results were potentially available to the
`colonoscopists. A clinical research organization
`(Parexel) received the results of Hemoccult II tests
`and colonoscopy directly from the clinical sites and
`received the results of fecal DNA analyses from the
`clinical laboratory (Exact Sciences).
`Parexel conducted the data analyses according
`to a prespecified plan and provided the results to
`the investigators after completion of the study. Only
`Parexel had access to the data until the blinding
`was removed, at which time the information was
`shared with the authors. The authors wrote the ar-
`ticle; Exact Sciences guaranteed the first author the
`right to publish the results of the study regardless
`of the outcome. Parexel, CareStat (the company that
`provided biostatistical support), and the authors
`each independently vouch for the veracity of the
`data and data analysis.
`
`study population
`The target population consisted of asymptomatic
`persons at average risk for colorectal cancer. The
`appropriate institutional review board at each site
`approved the study. Written informed consent was
`obtained from all participants. Study sites recruit-
`ed persons from local practices and undertook ac-
`
`*See NAPS document no. PC0001 for 112 pages of supplementary
`material regarding the study protocol. To order, contact NAPS, c/o
`Burrows Systems, P.O. Box 3976, New Hyde Park, NY 11040.
`
`n engl j med
`
`351;26
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`www.nejm.org december
`
`23, 2004
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`2705
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`The New England Journal of Medicine
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`Downloaded from nejm.org on October 23, 2023. For personal use only. No other uses without permission.
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` Copyright © 2004 Massachusetts Medical Society. All rights reserved.
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`
` new england journal
`The
`
` medicine
`of
`
`tivities to enhance the public’s awareness of colo-
`rectal cancer and the availability of screening. The
`costs of colonoscopy were not covered by the study;
`Hemoccult II and fecal DNA testing was provided
`without charge. Participants were compensated in
`a manner approved by each site’s institutional re-
`view board.
`All participants were at least 50 years old. En-
`rollment was stratified according to age, with a min-
`imum of three quarters of subjects 65 years of age
`or older. Exclusion criteria included gastrointesti-
`nal bleeding within the preceding month, a change
`in bowel habits or a recent onset of abdominal pain,
`previous colorectal cancer or polyps, prior resec-
`tion of any part of the colon, iron-deficiency ane-
`mia, or other coexisting visceral cancer. Persons who
`had undergone colonoscopy, sigmoidoscopy, or
`double-contrast barium enema within the preced-
`ing 10 years or who had had a positive fecal occult-
`blood test within the preceding 6 months were ex-
`cluded, as were those with inflammatory bowel
`disease, familial adenomatous polyposis or hered-
`itary nonpolyposis colon cancer, more than one first-
`degree relative with colorectal cancer, or any first-
`degree relative with colorectal cancer before the age
`of 50 years. Persons unwilling or unable to under-
`go colonoscopy were also excluded.
`
`procedures
`Subjects were given detailed instructions for stool
`collection; no dietary or medication modifications
`were required. Specimens were shipped directly
`to the clinical laboratory in a bar-coded container,
`chilled to between 0 and 4°C. Specimens were re-
`quired to arrive within 72 hours after collection; a
`minimal 30-g sample was required. If a sample
`failed to meet these requirements, another sample
`was sought before colonoscopy was performed.
`Samples were stored at –80°C until analysis.
`Subjects were given three Hemoccult II cards
`and instructions regarding dietary and medication
`modifications to comply with current recommen-
`1,19,20
`dations.
` Cards were returned to physicians’
`offices for non-rehydrated analysis by the physician
`or a designee, consistent with the manufacturer’s
`instructions and current guidelines. If all three cards
`(six panels) were not completed, additional cards
`were provided.
`Colonoscopy was performed with the prepara-
`tion and sedation customarily used at each site. The
`colonoscopist documented the extent of the colon
`that was visualized and the quality of the bowel
`
`preparation. Adequate colonoscopy required visu-
`alization of the cecum and a minimum of 90 per-
`cent of the mucosa. The size and location of any
`lesions were recorded. Biopsy and surgical resec-
`tion specimens were examined histopathologi-
`cally at each site; no centralized pathological review
`was performed.
`Subjects could be evaluated only if the speci-
`men for fecal DNA analysis was adequate, all six
`Hemoccult II panels had been completed, and co-
`lonoscopy was adequate. Subjects were classified
`according to the most advanced lesion identified.
`Advanced adenoma was defined as any lesion con-
`taining high-grade dysplasia, a polyp containing
`clinically significant villous architecture, or a tubu-
`lar adenoma that was at least 1 cm in diameter. Mi-
`nor polyps included tubular adenomas less than
`1 cm in diameter and hyperplastic polyps.
`Parexel provided the clinical laboratory with a
`coded list of stool specimens to be analyzed for
`DNA abnormalities on the basis of colonoscopy
`and pathological reports. The prespecified analytic
`plan was designed to maximize the study’s efficien-
`cy without compromising measures of sensitivi-
`ty, specificity, and adherence to the protocol. DNA
`analysis was performed on stool samples from all
`subjects with an invasive cancer or advanced ade-
`noma who could be evaluated and on randomly se-
`lected subgroups of 600 subjects with minor pol-
`yps and 1400 subjects with no polyps; these groups
`comprised the analyzed subgroup.
`
`fecal dna analysis
`All samples analyzed for fecal DNA were processed
`in a single laboratory. The fecal DNA panel con-
`ras
`sisted of 21 mutations: 3 in the K-
` gene, 10 in the
`APC
`p53
` gene, and 8 in the
` gene; the microsatellite-
`instability marker BAT-26; and a marker of long
`DNA thought to reflect disordered apoptosis of
`13,14,16
`cancer cells sloughed into the colonic lumen.
`The plan for DNA analyses has been described pre-
`13,16
` and is shown in Figure 1. Laboratory
`viously
`handling of all samples was fully automated, and
`quantitative analysis of the area under the curve, a
`measure of signal intensity of the labeled nucle-
`otides, was compared with that for control DNA
`fragments with a known mutation. Each marker
`was assessed independently; a positive result for any
`component of the panel constituted a positive fecal
`DNA test. Laboratory technicians were unaware of
`both the clinical data associated with each sample
`and the sampling protocol.
`
`2706
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`n engl j med
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`351;26
`
`www.nejm.org december
`
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`
`23
`
`2004
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`The New England Journal of Medicine
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`Downloaded from nejm.org on October 23, 2023. For personal use only. No other uses without permission.
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` Copyright © 2004 Massachusetts Medical Society. All rights reserved.
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`fecal dna for colorectal-cancer screening
`
`Whole-stool collection
`
`Homogenization
`
`Homogenate
`
`Centrifugation
`RNase treatment
`Concentration
`
`Crude DNA
`
`Use of “capture” probes
`Use of magnetic beads
`DNA elution
`
`Purified DNA
`(9 “captures” for K-ras, p53, APC, BAT-26;
`4 “captures” for long DNA)
`
`Mutation analysis
`
`Long-DNA analysis
`
`Use of biotinylated primers
`Use of Taq polymerase
`PCR amplification
`
`Use of PCR primers
`Use of probes
`Use of Taq polymerase
`
`Amplified gene targets
`(13 specific PCRs)
`
`Real-time PCR signal
`(16 real-time PCRs)
`
`Use of immobilization
`of PCR products
`Use of sequence-specific primers
`Use of labeled nucleotides
`
`Mutant-specific labeled products
`(22 mutant-specific reactions)
`
`Analysis of gene mutations
`and BAT-26
`
`Figure 1. Approach to Extraction and Analysis of Fecal DNA.
`Stool samples were thawed at room temperature and homogenized. Aliquots (each equivalent to 4 g of stool) were centri-
`fuged to remove particulate matter. Crude DNA was precipitated and resuspended. Sequence-specific DNA fragments were
`then purified from the total nucleic acid preparations by performing oligonucleotide-based hybrid “captures.” To analyze sam-
`ples directly for the presence of long DNA, the purified DNA was amplified in a real-time polymerase chain reaction (PCR), tar-
`geting fragments approximately 1.3, 1.8, and 2.4 kb from the capture site. In parallel, for the mutation portions of the assay,
`PCR amplification reactions were conducted with the use of biotinylated primers for specific gene targets. Each of the PCR
`products was then bound to magnetic-bead supports, and a mutation-specific, solid-phase minisequencing protocol was
`used to identify point mutations. BAT-26 deletions (of 4 to 15 bp) were identified according to the size of the reaction prod-
`ucts. All minisequencing reaction products were analyzed by capillary electrophoresis with laser-induced fluorescence detec-
`tion. Each marker was assessed independently; a positive result for any component of the panel constituted a positive fecal
`17
`DNA test.
`
`n engl j med
`
`351;26
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`www.nejm.org december
`
`23, 2004
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`2707
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` new england journal
`The
`
` medicine
`of
`
`statistical analysis
`The sample size was predetermined on the basis
`of the assumption that the fecal DNA panel and
`Hemoccult II had a sensitivity for the detection
`of colorectal cancer (i.e., tumor–node–metastasis
`[TNM] stage I through IV) of at least 65 percent and
`no more than 25 percent, respectively. Given this
`assumption, the enrollment of 32 subjects with co-
`lorectal cancer would provide the study with a sta-
`tistical power of 90 percent to detect a significant
`difference at a two-sided alpha level of 0.05 with the
`21
`use of McNemar’s test.
` A post hoc McNemar’s test
`was performed to compare the ability of the fecal
`DNA panel and Hemoccult II to identify subjects
`with fully specified advanced neoplasia (advanced
`adenoma or cancer). No interim analyses were per-
`formed, and missing data were not imputed.
`
`results
`
`study population
`A total of 5486 subjects were enrolled, of whom
`4404 could be fully evaluated; 1082 (19.7 percent)
`could not be evaluated (Fig. 2). The demographic
`and clinical characteristics of the population that
`could be evaluated and the subgroup that was ana-
`lyzed were similar (Table 1).
`Colonoscopic findings are shown in Table 2.
`Invasive adenocarcinoma was identified in 31 sub-
`jects (a prevalence of 0.7 percent). The higher prev-
`alence of pathological findings in the analyzed sub-
`group reflects the prespecified sampling strategy
`for stool processing in subjects with either no pol-
`yps or minor polyps. The only clinically significant
`complications were four colonoscopic perforations
`among 4404 subjects (0.09 percent).
`
`fecal dna panel versus hemoccult ii
`The fecal DNA panel detected 16 of 31 invasive
`cancers (TNM stage I, II, or III), for a sensitivity of
`51.6 percent; Hemoccult II detected 4 of 31 cancers,
`for a sensitivity of 12.9 percent (Table 2). The fecal
`DNA panel detected 13 cancers that were missed
`by Hemoccult II, whereas Hemoccult II detected
`1 cancer that was missed by the panel. This dif-
`ference in discordant test results was significant
`(P=0.003). In a post hoc analysis among subjects
`with node-negative disease (TNM stage I or II), the
`sensitivity of the fecal DNA panel was statistically
`superior to that of Hemoccult II (56.5 percent vs.
`13.0 percent, P=0.006). Among persons with TNM
`stage 0, I, II, or III (TNM 0 is carcinoma in situ), the
`
`fecal DNA panel had a sensitivity of 40.8 percent,
`whereas Hemoccult II had a sensitivity of 14.1 per-
`cent. The fecal DNA panel detected 22 lesions that
`were missed by Hemoccult II, whereas Hemoccult
`II detected 3 lesions missed by the panel. This dif-
`ference in discordant test results was significant
`(P<0.001).
`Among the 40 subjects who had adenomas
`with high-grade dysplasia, the fecal DNA panel de-
`tected 13 of the adenomas (32.5 percent), whereas
`Hemoccult II detected 6 (15.0 percent). For the de-
`tection of other advanced adenomas (villous polyps
`and tubular adenomas 1 cm in diameter or larger)
`and for minor polyps, the sensitivities of both tests
`were consistently less than 20 percent (Table 2).
`Among 418 subjects with advanced neoplasia (de-
`fined as a tubular adenoma 1 cm in diameter or
`larger, a polyp with a villous histologic appearance,
`a polyp with high-grade dysplasia, or cancer), the
`DNA panel was positive in 76 subjects, whereas
`Hemoccult II was positive in 45 subjects (18.2 per-
`cent vs. 10.8 percent, P=0.001). There was no sig-
`nificant difference in sensitivity according to the
`size of the cancer or advanced adenoma for either
`test (data not shown).
`Among 1423 subjects with negative findings
`on colonoscopy, 79 had a positive fecal DNA pan-
`el and 68 had a positive Hemoccult II test, for spec-
`ificities of 94.4 percent and 95.2 percent, respec-
`tively (Table 2). Among subjects with minor polyps,
`specificities for the fecal DNA panel and Hemoc-
`cult II were 92.4 percent and 95.2 percent, respec-
`tively.
`Table 3 shows the frequencies of abnormal com-
`ponents of the fecal DNA panel as they relate to the
`various histologic findings. All components of the
`panel contributed to the overall sensitivity of the
`test. Although no formal statistical analysis was per-
`formed because of the small size of the subgroups,
`APC, p53,
`the sensitivities of the point mutations in
`ras
`and K-
` were generally greater than those for the
`BAT-26 and long-DNA markers for clinically impor-
`tant lesions.
`
`discussion
`
`We compared a panel of fecal DNA markers and
`Hemoccult II as screening tests for colorectal can-
`cer in an average-risk, asymptomatic population.
`The sensitivity of the fecal DNA panel was four
`times that of Hemoccult II for invasive cancer and
`more than twice as sensitive for adenomas con-
`
`2708
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`n engl j med
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`351;26
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`www.nejm.org december
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`23
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`2004
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`fecal dna for colorectal-cancer screening
`
`with advanced adenomas were included in the analyzed subgroup.
`the analyzed subgroup. On subsequent audit of the data by the clinical research organization, these subjects were reclassified as having advanced adenomas. Thus, only 403 subjects
`cecum or that did not adequately visualize the colonic mucosa. Twenty-three subjects with advanced adenomas were originally classified as having minor polyps and were excluded from
`Some subjects had more than one reason for not being evaluated. A total of 641 subjects did not undergo colonoscopy; 129 subjects underwent a colonoscopy that did not reach the
`Figure 2. Disposition of Subjects Enrolled in the Study.
`
`2507 In analyzed subgroup
`
`for analysis
`
`1423 Randomly selected
`
`for analysis
`
`648 Randomly selected
`
`adenomas
`
`403 With advanced
`
`analyzed subgroup
`
`excluded from
`A total of 1897
`
`selected for analysis
`895 Randomly not
`
`selected for analysis
`979 Randomly not
`
`23 Excluded
`
`2318 With no polyps
`
`1627 With minor polyps
`
`adenomas
`
`426 With advanced
`
`cancer
`
`tumor, 1 with cloacogenic
`1 with a rectal carcinoid
`31 With adenocarcinoma,
`
`770 Did not complete colonoscopy
`426 Did not complete Hemoccult II cards
`641 Did not provide an adequate stool sample
`1082 Could not be evaluated
`
`4404 Could be fully evaluated
`
`written informed consent
`
`5486 Eligible subjects provided
`
`n engl j med
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`351;26
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`www.nejm.org december
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`23, 2004
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`2709
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` new england journal
`The
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` medicine
`of
`
`Table 1. Characteristics of Subjects Who Could Be
`Evaluated and Those Who Were Analyzed.
`
`Group That
`Could Be
`Evaluated
`(N=4404)
`
`Analyzed
`Subgroup
`(N=2507)
`
`68.6
`570 (12.9)
`1971 (44.8)
`1678 (38.1)
`185 (4.2)
`1963(44.6)
`
`69.5
`210 (8.4)
`1150 (45.9)
`1025 (40.9)
`122 (4.9)
`1115 (44.5)
`
`3846 (87.3)
`369 (8.4)
`189 (4.3)
`615 (14.0)
`
`2180 (87.0)
`217 (8.7)
`110 (4.4)
`348 (13.9)
`
`Characteristic
`
`Age
`Mean — yr
`50–59 yr — no. (%)
`60–69 yr — no. (%)
`70–79 yr — no. (%)
`≥80 yr — no. (%)
`Male sex — no. (%)
`Race or ethnic group
`— no. (%)
`White
`Black
`Other
`Family history of colorectal
`cancer — no. (%)
`
`taining high-grade dysplasia. This increase in sen-
`sitivity was achieved without a loss of specificity
`among persons with no polyps on colonoscopy. Al-
`though this study was not powered to compare
`the tests among the different stages of cancer, the
`fecal DNA panel appears to be more sensitive than
`Hemoccult II for the detection of early (TNM stage
`I or II) colorectal cancer. However, since this result
`was not prespecified in the analytic plan, it should
`be considered preliminary.
`The sensitivity of the fecal DNA panel for the de-
`tection of cancer was lower than that in previous
`13,14,16,17,22,23
`reports.
` Using a similar panel, Ahl-
`quist and colleagues reported a sensitivity of 90
`percent for cancer and 82 percent for advanced ad-
`13
` They used archived stool
`enomatous polyps.
`specimens from patients with known cancer, many
`of whom had advanced disease. The differences
`in test characteristics between their study and ours
`may be explained by differences in the clinical spec-
`trum of disease (e.g., tumor size, tumor stage, or
`location within the colon) and study methods.
`Tagore et al. used an identical panel of markers to
`analyze stool specimens from 52 patients with co-
`lorectal cancer and 28 patients with advanced ad-
`enomas and reported sensitivities of 64 percent
`16
`and 57 percent, respectively.
` Despite the predom-
`inance of distal lesions in that study, the test char-
`acteristics are closer to those in our study. In other
`
`studies, with the use of different panels of mark-
`ers or different techniques, the sensitivity for the
`detection of cancer has ranged from 37 percent to
`14,17,22,23
`71 percent.
` However, none of these stud-
`ies examined asymptomatic persons exclusively.
`After we designed our protocol, the detection of
`“advanced neoplasia” became a popular outcome
`measure for studies involving screening for colorec-
`24-28
`tal cancer.
` This term includes more advanced
`polyps with respect to size (tubular adenomas 1 cm
`in diameter or larger), histologic findings (villous
`architecture or high-grade dysplasia), or both fea-
`tures, along with invasive cancer. We compared
`the sensitivity of the fecal DNA panel with that of
`Hemoccult II in a post hoc analysis of the detection
`of all cases of advanced colorectal neoplasia, which
`included cancers and advanced adenomas. The fecal
`DNA panel detected 18.2 percent of samples with
`advanced neoplasia, whereas Hemoccult II identi-
`fied 10.8 percent. The sensitivity of the DNA panel
`for advanced adenomas was lower than previously
`15-17,29
` although the confidence intervals
`reported,
`overlap those in other reports. A plausible explana-
`tion for this difference in sensitivity is a decrease in
`exfoliation of cells owing to smaller adenoma size,
`since our subjects had smaller advanced adenomas
`than those in other studies (data not shown).
`The sensitivity of Hemoccult II — 13 percent
`for the detection of cancer — is lower than that re-
`25,30,31
`ported in other series.
` Our finding should
`most appropriately be compared with the results
`of previous studies in which a reference standard
`such as colonoscopy was used in all subjects, irre-
`spective of the results of the fecal occult-blood test.
`The most relevant study reported a sensitivity for
`cancer of 21 percent among 1217 subjects who were
`undergoing surveillance colonoscopy after curative
`resection of a colorectal neoplasm; these results
`30
` Lieberman and
`are consistent with our findings.
`Weiss identified 12 of 24 patients with invasive can-
`25
`cer using rehydrated samples for Hemoccult II.
`We did not rehydrate the Hemoccult II cards, an
`approach that is consistent with published guide-
`2,19,20
`lines.
` Our sensitivity results for Hemoccult
`II may more closely reflect its sensitivity in clinical
`practice; the difference between our results and
`those of other reports is potentially important and
`deserves further study.
`An advantage of using DNA as the analyte is that
`a marker panel can be expanded or refined as knowl-
`edge about tumor biology evolves. It is worth not-
`ing that in our study the sensitivity of the specific
`
`2710
`
`n engl j med
`
`351;26
`
`www.nejm.org december
`
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`
`23
`
`2004
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`The New England Journal of Medicine
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`Downloaded from nejm.org on October 23, 2023. For personal use only. No other uses without permission.
`
` Copyright © 2004 Massachusetts Medical Society. All rights reserved.
`
`Geneoscopy Exhibit 1042, Page 7
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`fecal dna for colorectal-cancer screening
`
`Table 2. Most Advanced Finding at Colonoscopy and Results of the Fecal DNA Panel and Occult-Blood Test in the Analyzed Subgroup.*
`
`Most Advanced Finding
`at Colonoscopy
`
`Adenocarcinoma
`TNM stage I
`TNM stage II
`TNM stage III
`TNM stage IV
`Adenocarcinoma + high-grade
`dysplasia
`Advanced adenoma
`High-grade dysplasia
`Villous adenoma
`Tubular adenoma ≥1 cm
`Unspecified
`Minor polyps‡
`Tubular adenoma <1 cm
`Hyperplastic
`Unspecified
`No polyps on colonoscopy§
`
`Group That
`Could Be
`Evaluated
`(N=4404)
`
`Analyzed
`Subgroup
`(N=2507)†
`
`no.
`
`31
`15
`8
`8
`0
`72
`
`426
`41
`139
`230
`16
`1627
`762
`633
`232
`2318
`
`31
`15
`8
`8
`0
`71
`
`403
`40
`133
`214
`16
`648
`286
`276
`86
`1423
`
`Positive Fecal DNA Panel
`
`Positive Occult-Blood Test
`
`no./total no.
`16/31
`8/15
`5/8
`3/8
`0
`29/71
`
`61/403
`13/40
`24/133
`23/214
`1/16
`49/648
`23/286
`17/276
`9/86
`79/1423
`
`% (95% CI)
`51.6 (34.8–68.0)
`53.3 (30.1–75.2)
`62.5 (30.6–86.3)
` 37.5 (13.7–69.4)
`
`40.8 (30.2–52.5)
`
` 15.1 (12.0–19.0)
`32.5 (20.1–48.0)
`18.0 (12.4–25.4)
`10.7 (7.3–15.6)
`6.2 (1.1–28.3)
`7.6 (5.8–9.9)
`8.0 (5.9–12.7)
`6.2 (3.9–9.6)
`10.5 (5.6–18.7)
`5.6 (4.5–6.9)
`
`no./total no.
`4/31
`1/15
`2/8
`1/8
`0
`10/71
`
`43/403
`6/40
`13/133
`22/214
`2/16
`31/648
`15/286
`10/276
`4/86
`68/1423
`
`% (95% CI)
`12.9 (5.1–28.9)
`6.7 (1.2–29.8)
`25.0 (7.1–59.1)
`12.5 (2.2–47.1)
`
` 14.1 (7.8–24.6)
`
`10.7 (8.0–14.1)
`15.0 (7.1–29.1)
`9.8 (5.8–16.0)
`10.3 (6.9–15.1)
` 12.5 (3.5–36.0)
`4.8 (3.4–6.7)
`5.2 (3.5–9.2)
`3.6 (2.0–6.5)
`4.6 (1.8–11.4)
`4.8 (3.9–5.8)
`
`* The total in both the group that could be evaluated and the analyzed subgroup includes two subjects who are not included in any other cate-
`gory in the table; one had a rectal carcinoid, and one had cloacogenic cancer. The subject with rectal carcinoid was not identified by means of
`either fecal DNA or by fecal occult-blood testing. The subject with cloacogenic cancer was identified by means of fecal DNA testing, but not
`by fecal occult-blood testing. CI denotes confidence interval, and TNM tumor–node–metastasis.
`† Stool specimens were selected for DNA testing on the basis of available data (i.e., polyp size and histologic findings) at the time of selection
`for processing. Subsequent audit of data by the clinical research organization resulted in reclassification of less than 5 percent of subjects.
`‡ The fecal DNA panel had a specificity of 92.4 percent, and the occult-blood test had a specificity of 95.2 percent (95 percent confidence interval
`for the difference in specificity, ¡5.4 percent to 0.1 percent).
`§ The fecal DNA panel had a specificity of 94.4 percent, and the occult-blood test had a specificity of 95.2 percent (95 percent confidence interval
`for the difference in specificity, ¡2.4 percent to 0.9 percent).
`
`gene-mutation components of the panel for the de-
`tection of cancer was similar to that in previous
`13,16,22
`studies involving symptomatic patients.
` The
`sensitivity of the long-DNA assay component was
`lower than expected, a finding that may be related to
`DNA degradation. This first-generation assay panel
`has already been improved by enhanced techniques
`32
`for extraction of human DNA from stool.
`Our study has certain limitations. First, persons
`65 years of age and over were disproportionately
`represented in the study population. However, there
`is no reason to expect the distribution of DNA ab-
`normalities to vary according to age; the observed
`sensitivity is likely to apply to younger populations
`with a lower prevalence of advanced neoplasia. Sec-
`ond, the study was designed as a direct comparison
`
`of two noninvasive screening methods; there were
`too few cancers and advanced adenomas with high-
`grade dysplasia to provide narrow confidence in-
`tervals for the estimated sensitivity of either test.
`Third, no inference can be drawn about the ap-
`propriate interval for retesting or the effectiveness
`of repeated testing with the fecal DNA panel. Fourth,
`whereas all DNA analyses of samples were per-
`formed in a single laboratory with extensive expe-
`rience in the use of the assay, widespread use could
`introduce variability that would affect the sensitivi-
`ty and specificity.
`The place of fecal DNA testing in the current
`scheme of colorectal-cancer screening is beyond
`the scope of this discussion. The issue requires con-
`sideration of the characteristics of the test, risk,
`
`n engl j med
`
`351;26
`
`www.nejm.org december
`
`23, 2004
`
`2711
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`The New England Journal of Medicine
`
`Downloaded from nejm.org on October 23, 2023. For personal use only. No other us