ORIGINAL ARTICLE
`
`DNA Stabilization Is Critical for Maximizing Performance
`of Fecal DNA-Based Colorectal Cancer Tests
`
`Jeff Olson, BS, Duncan H. Whitney, PhD, Kristine Durkee, PhD, and Anthony P. Shuber, MS
`
`Abstract: We have developed a multitarget, fecal DNA screening
`assay that detects the presence of gene-specific mutations and long
`DNA fragments associated with colorectal cancer (CRC). We con-
`tinue to investigate methods that may be used to optimize clinical
`sensitivity. The goals of this investigation are to establish how sample
`handling conditions affect the stability of DNA in stool, thereby
`potentially limiting clinical sensitivity, and to determine conditions to
`ameliorate DNA degradation. A study was run comparing paired sam-
`ple aliquots. Quantitative PCR data for matched aliquots was used to
`determine first the effect of sample incubation on total recovery and
`integrity of DNA, then the effect of stabilization buffer addition to
`stool on recoverable DNA, and finally the impact of buffer addition
`on assay sensitivity. Comparison of quantitative PCR data for paired
`aliquots shows that the amount of recoverable human DNA is nega-
`tively affected by storing stool samples (N = 43) at room temperature
`for $36 hours (P = 0.0018). However, the addition of stabilization
`buffer leads to a significant increase in recovery of DNA (P = 0.010),
`compared with samples incubated without buffer. Whereas the DNA
`Integrity Assay (DIA) is found to be sensitive to DNA degradation
`(sensitivity was reduced by 82%; P = 0.0002), point mutation marker
`sensitivity is more refractory. Overall, DNA can be stabilized by
`addition of buffer to the sample, leading to increased assay sensitivity.
`
`Key Words: colorectal cancer, multitarget assay, DNA integrity,
`cancer screening, stool DNA
`
`(Diagn Mol Pathol 2005;14:183–191)
`
`C olorectal cancer (CRC) is the fourth most prevalent
`
`cancer in the United States and is the second leading
`cause of cancer deaths.1 More than 90% of colorectal cancer
`cases could be cured if detected in its earliest stages.2 Current
`colorectal cancer screening guidelines include a variety of op-
`tions including fecal occult blood test (FOBT), flexible sigmoid-
`oscopy, double-contrast barium enema, and colonoscopy.1–3
`Whereas being the most sensitive,4 the financial costs, man-
`power requirements, and potential complications associated
`with colonoscopy present formidable obstacles to its imple-
`mentation for large-scale, nationwide CRC screening.5 The
`other methods are less sensitive and are either invasive, or in
`the case of FOBT, depend upon a nonspecific, indirect as-
`sessment of blood in fecal matter. Fecal DNA methods have
`
`From Exact Sciences Corporation, Marlborough, MA.
`Reprints: Anthony P. Shuber, Chief Technology Officer, Exact Sciences
`Corporation, 100 Campus Drive, Marlborough, MA 01752 (e-mail:
`dwhitney@exactsciences.com).
`Copyright Ó 2005 by Lippincott Williams & Wilkins
`
`Diagn Mol Pathol  Volume 14, Number 3, September 2005
`
`been developed that are noninvasive and present continued
`opportunity for improvement as new molecular markers asso-
`ciated with CRC are identified and as new DNA detection tech-
`nologies are developed. Results of several targeted studies to
`assess sensitivity and specificity of fecal DNA tests have been
`previously reported,6–9 with sensitivities ranging from 52% to
`91% and specificities of 93% to 98%. Although these studies
`offer a confirmation of the potential benefits of fecal DNA
`screening protocols, it is known that several variables can affect
`test performance. Markers must be chosen that yield an ac-
`ceptable clinical sensitivity for the intended application (ie,
`screening average-risk individuals for sporadic disease). The
`fecal DNA assay is based on a combination of a panel of point
`mutations in APC, p53, and Kras genes, as well as a micro-
`satellite instability (MSI) marker, BAT-26, and a marker for
`long DNA fragments, DNA integrity assay (DIA). Addition-
`ally, mutation detection methods must be chosen that offer
`sufficient analytical sensitivity because the human DNA re-
`covered from stool is highly heterogeneous. Normal cells are
`sloughed into the colonic lumen along with the mutant cells.
`Therefore, analytical methods must be chosen that can detect
`as little as 1% mutant DNA in the presence of excess wild-type
`DNA. Also, sample prep methodologies must be chosen that
`allow for maximum recovery of human DNA from samples.
`Most of the DNA recovered from stool is bacterial in origin,
`with the human DNA component representing only a small
`minority. Purification methodologies must be able to effi-
`ciently select for the rare human component, and because the
`mutant copies (when they exist) represent only a small
`percentage of the total human DNA from stool, it is important
`to maximize the recovery of human DNA to maximize the
`probability of amplifying mutant copies in the PCR reactions.
`Development of a new affinity gel electrophoresis method that
`meets these needs has recently been described.10 Lastly, it is
`imperative to preserve the DNA in stool, such that it does not
`degrade during sample handling. A common method to ensure
`that DNA remains stable is to freeze stool samples as quickly
`as possible after collection or to receive samples in centralized
`testing labs as quickly as possible. However, to provide the
`option of decentralized sample analysis and still
`retain
`maximum sample integrity, it is desirable to develop a more
`robust and standardized sample-handling method.
`A multicenter study was recently completed to evaluate
`the sensitivity of a multitarget fecal DNA assay relative to
`FOBT in an average-risk population.9 The study had 31 can-
`cers, confirmed through colonoscopy, by screening approxi-
`mately 5,000 patients, and the majority of cancers were found
`to represent early-stage disease. Even though the study dem-
`onstrated a 4-fold greater sensitivity than FOBT, the fecal
`
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`Olson et al
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`Diagn Mol Pathol  Volume 14, Number 3, September 2005
`
`DNA assay was expected to detect a greater proportion of the
`cancers. The sensitivity contributed by the point mutation
`panel of markers was found to be consistent with previous
`studies,6–8 but the DIA portion of the test contributed sig-
`nificantly lower sensitivity to the overall assay than what had
`been seen previously, raising the question of how sample han-
`dling may affect DNA stability, in general, and the sensitivity
`of the different parts of the multitarget assay, more specifically.
`With the possibility that DNA degradation might lead
`to loss of marker sensitivity and overall assay performance,
`methods of making sample collection and handling more ro-
`bust were considered. Here we present experimental results
`that not only demonstrate how sample handling can affect
`DNA stability but also how degradation can be ameliorated by
`addition of buffer to stool samples shortly after collection.
`
`MATERIALS AND METHODS
`
`Sample Collection and Incubation
`A total of 43 samples were collected from known CRC
`patients as well as patients without cancer by a separate orga-
`nization (Indivumed GmbH, Hamburg, Germany) that also
`managed all patient informed consent and compliance with
`human subject guidelines. All stool samples were frozen within
`1 hour of defecation and shipped to EXACT Sciences on dry
`ice (278°C). Once received, samples were subjected to pre-
`scribed room temperature incubation times as described later.
`Prior to the start of the incubation time course, stool samples
`were thawed and 1 aliquot was processed to recover DNA
`immediately (t0). The DNA from the t0 aliquot for all samples
`was analyzed and served as an incubation control. The
`remainder of the stool was left to incubate at room tem-
`perature. At prescribed time points, aliquots were removed
`from the stool and human DNA was recovered and analyzed in
`similar manner to the controls.
`All aliquots were standardized by weight (30 g). Experi-
`ments were designed to measure the effect of incubation time
`on DNA integrity, as well as the quantity of recoverable DNA.
`The experiments also included an addition of stabilization buffer
`to stool aliquots. Stabilization buffer consisted of 0.5 mol/L
`Tris, 0.15 mol/L EDTA, and 10 mmol/L NaCl (pH 9.0). In
`these experiments, aliquots were stored at room temperature
`for 36 or 48 hours, with and without buffer added. In the case
`of buffer addition, the buffer was simply added to the stool
`aliquot in a plastic container with a lid, but no effort was made
`to homogenize the sample. At the prescribed time period, the
`aliquots with and without buffer were processed to recover
`human DNA and then analyzed by the DIA assay. An
`additional set of experiments was conducted to study the effect
`of incubation time on specific gene mutations. In this experi-
`ment, 1 set of samples (6 samples) was incubated for 36, 48, or
`72 hours without any stabilization buffer added. Another set of
`samples (5 samples) was incubated for 36 or 48 hours, with
`and without buffer added. After the prescribed incubation
`time, all samples were processed to recover and purify human
`DNA and analyze the DNA for gene mutations as described
`later.
`
`184
`
`Recovery of DNA From Stool
`The sample preparation methodology used to recover
`DNA from stool was previously reported.6,10 Stool aliquots
`were weighed and combined with Exact buffer A (1:7 (w/v)
`ratio) and homogenized on an Exactor (Exact Sciences). After
`homogenization, a 4-g stool equivalent (;32 mL) of each
`sample was centrifuged to remove all particulate matter. The
`supernatants were then treated with 20 mL TE buffer (0.01 mol/L
`Tris [pH 7.4] and 0.001 mol/L EDTA) containing RNase A
`(2.5 mg/mL) and incubated at 37°C for 1 hour. Total nucleic
`acid was then precipitated (first adding 1/10 volume 3 mol/L
`NaAc, then an equal volume of isopropanol). Genomic DNA
`was pelleted by centrifugation, the supernatant removed, and
`the DNA resuspended in TE.
`
`Human DNA Purification
`Target human DNA fragments were purified from total
`nucleic acid preparations using a newly developed DNA
`affinity electrophoresis purification methodology. This method
`has recently been described in detail.10 In brief, human DNA
`can be separated from the excess bacterial DNA by hybridi-
`zation of the target sequences to complementary, covalently
`bound oligonucleotide capture probes in acrylamide gel mem-
`branes. Crude human DNA preparations (2,400 mL) were mixed
`with 960 mL formamide (Sigma), 385 mL 103 TBE, and
`filtered through a 0.8-mm syringe filter (Nalgene, Rochester, NY)
`and then denatured (heated at 95°C for 10 minutes, then
`cooled in ice for 5 minutes). The sample mix was loaded on
`top of the capture membrane, and electrodes above and below
`the capture layer were applied. Samples were electrophoresed
`(15 V, 16 hours) using TBE in the reservoirs above and below
`the capture layer. After electrophoretic capture, the remaining
`solution was removed from the tubes, and the tube array was
`separated from the capture plate. The capture membranes were
`then washed and 40 mL of 100 mmol/L NaOH was added to
`the top of the capture membrane and incubated for 15 minutes.
`The capture plate was placed on top of a custom molded 48-
`well DNA collection plate and centrifuged briefly (1,900 3 g)
`to recover the eluted DNA. Then, 8 mL of neutralization buffer
`(500 mmol/L HCL + 0.13 TE) was added to each well of the
`collection plate and mixed.
`
`Quantification of Recovered Human DNA by
`TaqMan Analysis
`TaqMan analysis was performed on an I-Cycler
`(BioRad) with primers against a 200-bp region of the APC
`gene. A probe labeled with 6-carboxyfluorescein (FAM) and
`6-carboxytetramethylrhodamine (TAMRA) was used to detect
`PCR product. Amplification reactions consisted of captured
`human stool DNA mixed with 103 PCR buffer, LATaq en-
`zyme (Takara), 13 PCR primers (5 mmol/L), and 13 TaqMan
`probe (2 mmol/L; Biosearch Technologies). We used 5 mL of
`captured DNA in the PCR reactions. TaqMan reactions were
`performed with the same program as described below (DIA).
`
`Sequence-Specific Amplification
`Polymerase chain reaction (PCR) amplifications (50 mL)
`were performed on MJ Research Tetrad Cyclers (Watertown,
`MA) using 10 mL of purified DNA, 103 PCR buffer
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`Diagn Mol Pathol  Volume 14, Number 3, September 2005
`
`DNA Stabilization
`
`(Takara Bio Inc, Madison, WI), 0.2 mmol/L dNTPs (Promega,
`Madison, WI), 0.5 mmol/L sequence-specific primers (Mid-
`land Certified Reagent Co, Midland, TX), and 2.5 U LATaq
`DNA polymerase (Takara). All amplification reactions were
`performed under identical thermocycler conditions: 94°C for
`5 minutes and 40 cycles consisting of 94°C (1 minutes.), 60°C
`(1 minute), and 72°C (1 minute), with a final extension of
`5 minutes at 72°C. Thirteen separate PCR reactions were run per
`sample. For analysis of each of the PCR products, 8 mL of each
`amplification reaction was loaded and electrophoresed on a
`4% ethidium bromide–stained NuSieve 3:1 agarose gel (FMC,
`Rockland, ME) and visualized with a Stratagene EagleEye II
`(Stratagene, La Jolla, CA) still image system. All oligonucle-
`otide sequences (capture probes, PCR primers, and TaqMan
`probes) are available upon request.
`The multitarget assay was designed to have 13 separate
`PCR reactions in the multiple mutation (MuMu) panel and 12
`PCR reactions in the DIA portion of the assay.
`
`Mutation Panel Analysis
`The presence or absence of point mutations or Bat-26–
`associated deletions was determined by using modified solid-
`phase single-base extension (SBE) reactions. Point mutation
`targets included codons K12p1, K12p2, and K13p2 on the K-
`ras gene; codons 876, 1306, 1309, 1312, 1367p1, 1378p1,
`1379, 1450p1, 1465 and 1554 on the APC gene; and codons
`175p2, 245p1, 245p2, 248p1, 248p2, 273p1, 273p2, and
`282p1 on the p53 gene. Including the Bat-26 deletion marker,
`the panel consisted of 22 markers in total. For all gene targets,
`separate wild-type and mutant specific reactions were per-
`formed. Details of the reactions and analysis using capillary
`electrophoresis have been previously described.10
`
`DIA
`
`The DIA assay has been previously described in detail.11
`More recently, this assay has been converted to a real-time
`PCR methodology (unpublished data). Three unique PCR re-
`actions (in duplicate) per loci were run on I-Cycler instruments
`(BioRad, Hercules, CA). The strategy was to capture locus-
`specific segments and perform small (;100 bp) PCR amplif-
`ications remote from the capture site as an indicator of DNA
`length. DNA fragments for integrity analysis were amplified
`from 4 different loci: 17p13, 5q21, HRMT1L1, and LOC91199.
`PCR primer sets and associated TaqMan probe for each loci
`of interest are ‘‘walked’’ down the sequence, thereby inter-
`rogating for the presence and quantity of increasing length of
`DNA of approximately 1,300 bp, 1,800 bp, and 2,400 bp
`fragments of captured DNA. Purified DNA template (5 mL)
`was mixed with 5 mL 103 PCR buffer (Takara), 10 mL dNTPs
`(2 mmol/L, Promega), 0.25 mL LATaq (5 U/mL, Takara),
`24.75 mL molecular biology grade water (Sigma), and 5 mL
`of a mix of PCR primers (5 mmol/L, Midland) and TaqMan
`dual-labeled probes (2 mmol/L, Biosearch Technologies). The
`I-Cycler was programed as follows: 94°C for 5 minutes and
`then 40 cycles of 94°C for 1 minute, 55°C for 1 minute, and
`72°C for 1 minute. Genomic standards, prepared as 20, 100,
`500, 2,500, and 12,500 GE/5 mL, were prepared and used to
`generate a standard curve.
`
`q 2005 Lippincott Williams & Wilkins
`
`DIA Data Analysis
`Threshold genome equivalents (GE) values were de-
`termined for each of 12 PCR reactions (corresponding to the
`1.3kb, 1.8kb, and 2.4kb fragments across the 4 genomic loci)
`using a previously determined set of cancers and normals. We
`then applied a requirement that at least 4 of the 12 PCR re-
`actions are above the individual PCR thresholds to prospec-
`tively determine cancers.
`
`Statistical Methods
`The impact of sample incubation on DNA recovery and
`the impact of stabilization buffer on DNA recovery were both
`assessed using quantitative PCR analysis. The data for both
`comparisons were subjected to a paired-sample t test using
`GraphPad QuickCalcs software (GraphPad Software, Inc, San
`Diego, CA). The effect of sample incubation on the integrity of
`recoverable DNA was analyzed using DIA cutoffs. Resulting
`DIA scores after incubation were analyzed by a Fisher exact
`test compared with controls that had been analyzed at t0, using
`GraphPad QuickCalcs.
`
`RESULTS
`
`Effect of Sample-Handling Conditions on DIA
`DIA is a measure of long DNA which has been shown to
`be an independent and effective marker of CRC.11 In these
`experiments,
`the effect of sample-handling conditions on
`recoverable DNA is assessed using DIA. Moreover, the results
`of the DIA analysis are a direct indication of how this marker is
`impacted under the prescribed conditions and likewise how
`CRC detection sensitivity is affected. A total of 38 samples
`were analyzed by DIA. Twenty-seven were DIA negative at t0,
`and aliquots were incubated with and without stabilization
`buffer. There was no significant amount of long DNA present
`in these samples to judge the effect of incubation conditions on
`DNA stability. However,
`impact of sample-handling con-
`ditions was assessed by total recoverable DNA (ie, 200 bp
`results). A sampling of results from this group of specimens is
`shown in Table 1. A DNA recovery score was calculated by
`averaging the results for the 4 separate loci (D, E, X, and Y) for
`each sample. Without any buffer added,
`the majority of
`samples (18/27, 67%) stored at room temperature ($36 hours)
`yielded less than 50% of the DNA recovered at
`t0. The
`remaining 9 samples (33%) had mild loss of recovery, yielding
`equivalent DNA, or less than a 50% loss, upon room tem-
`perature incubation. When aliquots were incubated with
`buffer, 81% (22/27) of the DIA-negative samples were pre-
`served (samples GP30, GP33, and GP96, in Table 1, are shown
`as examples). In the remaining 5 DIA-negative samples, the
`addition of stabilization buffer did not offer any significant
`advantage in recoverable DNA, compared with samples stored
`without buffer (see for example, LSP20-21 in Table 1).
`Of the 38 samples analyzed by DIA, 11 were found to be
`positive at t0. Table 2 shows the detected copy numbers of each
`DIA marker for these samples at t0 and extended incubation
`times, with and without stabilization buffer added. The DIA
`score (number of positive markers per sample) is also indi-
`cated. When no stabilization buffer
`is added, DNA is
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`Olson et al
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`Diagn Mol Pathol  Volume 14, Number 3, September 2005
`
`TABLE 1. Quantification of Recoverable Human DNA from Selected DIA-Negative Samples Incubated With and Without
`Stabilization Buffer
`
`Sample ID
`
`Incubation Conditions
`
`D200 (GE/10 mL)
`
`E200 (GE/10 mL)
`
`X200 (GE/10 mL)
`
`Y200 (GE/10 mL)
`
`GP30
`
`GP96
`
`GP33
`
`LSP20-21
`
`Fresh
`36 h no buffer
`36 h with buffer
`Fresh
`36 h no buffer
`36 h with buffer
`Fresh
`36 h no buffer
`36 h with buffer
`Fresh
`36 h no buffer
`36 h with buffer
`
`247
`50
`219
`129
`63
`34
`3,690
`61
`2,220
`1,140
`219
`542
`
`196
`27
`216
`1,120
`130
`1,270
`13,200
`136
`4,640
`807
`588
`363
`
`418
`76
`390
`604
`100
`1,140
`23,300
`422
`3,860
`1,500
`421
`174
`
`129
`0
`172
`769
`100
`1,290
`0
`103
`2
`337
`285
`591
`
`significantly degraded in 9 of the 11 originally DIA-positive
`stool samples when stored for $36 hours at room temperature.
`These would therefore be graded as DIA negative after room
`temperature incubation, resulting in an 82% loss in sensitivity
`(P = 0.002) for DIA. However, for these same samples, ad-
`dition of stabilization buffer prior to room temperature incu-
`bation yields significantly higher DNA copy number, such that
`all of the samples (11/11) would remain DIA positive, even
`after room-temperature incubation. Two of the samples (GP-031
`and GP-079) yielded high quantities of long DNA fragments
`even upon extended room temperature incubation without any
`added stabilization buffer.
`
`Effect of Sample Handling Conditions on Gene
`Mutation Markers
`It has previously been shown that DNA recovered from
`stool can be interrogated for specific mutations known to be
`associated with CRC.6–8 In the experiments described earlier, it
`was shown that upon room temperature incubation of stool
`samples long fragments of DNA are degraded, significantly
`diminishing the usefulness of the DIA markers. Further it was
`shown that human DNA yield is reduced introducing the ques-
`tion of whether or not sufficient amount of DNA template
`molecules remain for point mutation analysis in known CRC
`associated genes (eg, Kras, APC, and p53). Stool samples
`from 11 confirmed CRC patients were collected and shown to
`contain 1 or more point mutations, as summarized in Table 3.
`The amount of DNA recovered from aliquots stored
`under the different conditions is based on quantification of
`a 200-bp DNA fragment (Table 3). Although the amount of
`recoverable DNA varies widely from sample to sample, at t0
`the average recovery was 14,891 copies/10 mL, whereas after
`room temperature incubation, the average recovery was 1,955.
`Without buffer added to the samples, between 65% to 98% of
`the DNA was no longer recoverable (excluding sample GP-031),
`after incubation of samples at room temperature, compared
`with t0. The one exception, sample GP-031, maintained high
`DNA yield even without addition of stabilization buffer.
`Samples incubated with stabilization buffer maintained human
`DNA yields similar to the t0 samples (Table 3).
`
`186
`
`Aliquots from all samples were analyzed for mutations
`initially (t0), and additional aliquots were analyzed after room
`temperature incubation. Aliquots from the first 6 stool samples
`(Table 3) were simply stored at room temperature with no
`buffer added, whereas aliquots from the next set of 5 samples
`were stored with and without the addition of stabilization
`buffer. Mutations were reproducibly detected in 10 of the 11
`samples. Sample GP-105 was originally shown to contain an
`APC mutation (at codon 1554), and after incubation of an
`aliquot for 48 hours without buffer, the mutation was no longer
`detectable even with repeated analysis. However, when GP-
`105 was incubated in the presence of stabilization buffer, the
`originally identified mutation was detected. In addition, as we
`observed within the DIA marker experiments, human DNA
`recovery for all samples remained high when incubated in the
`presence of stabilization buffer, and DNA recovered from
`these aliquots also maintained detectable mutations.
`
`DISCUSSION
`The ability to recover human DNA from stool samples
`and identify mutations associated with colorectal cancer has
`been shown by several groups over the last decade. Sidransky
`et al12 first reported interrogating K-ras mutations associated
`with sporadic CRC in stool DNA in 1992. Subsequent reports
`also involved interrogation of single genetic targets.13–17 More
`recently, assays with multiple markers have been developed18,19
`to yield increased assay sensitivity in light of the genetic het-
`erogeneity of sporadic CRC cases. Furthermore, an assay for
`long DNA recovered from stool has been developed and
`shown to be associated with CRC with high specificity.11 The
`DIA itself was shown to detect 57% of cancers in one study11
`and has been shown to detect from 37%8 to 67%6 of cancers
`when incorporated in a multitarget assay. The multitarget tests
`have the potential to be used in population-based screening
`applications. However, in all cases, one of the central chal-
`lenges is to preserve the integrity of human DNA in the hostile
`stool environment, particularly during sample transport, to
`recover, amplify, and interrogate the DNA for known cancer-
`related abnormalities. Nucleases that are active in stool have
`the potential to rapidly degrade DNA, including the minor human
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`Diagn Mol Pathol  Volume 14, Number 3, September 2005
`
`DNA Stabilization
`
`TABLE 2. Results of DNA Integrity Assay for DIA-positive Samples Incubated at Room Temperature for Prescribed Times, With and
`Without Stabilization Buffer. DIA Scores (A) and Total Recoverable DNA (B) are Shown for 11 Samples Found to be DIA-positive
`at t0
`
`DIA-D
`
`DIA-E
`
`DIA-X
`
`DIA-Y
`
`(A)
`
`Sample
`
`Time Point
`
`1.3KB 1.8KB 2.4KB 1.3KB 1.8KB 2.4KB 1.3KB 1.8KB 2.4KB 1.3KB 1.8KB 2.4KB LcDIA Score
`
`GP32
`
`GP29
`
`GP34
`
`GP38
`
`GP31
`
`107
`
`117
`
`97
`
`105
`
`90
`
`GP79
`
`Sample
`
`GP32
`
`GP29
`
`GP34
`
`0 HR
`36 HR NB
`36 HR B
`0 HR
`24 HR NB
`24 HR B
`36 HR NB
`36 HR B
`0 HR
`24 HR NB
`24 HR B
`36 HR NB
`36 HR B
`0 HR
`36 HR NB
`36 HR B
`0 HR
`36 HR NB
`36 HR B
`0 HR
`48 HR
`48 HR, Buffer
`0 HR
`48 HR
`48 HR, Buffer
`0 HR
`48 HR
`48 HR, Buffer
`0 HR
`48 HR
`48 HR, Buffer
`0 HR
`48 HR
`48 HR, Buffer
`0 HR
`48 HR
`48 HR, Buffer
`
`15
`0
`55
`394
`0
`812
`0
`10
`1180
`10
`6170
`0
`1250
`735
`0
`88
`200
`274
`129
`672
`0
`567
`170
`0
`1170
`36
`0
`50
`71
`0
`29
`469
`0
`1320
`250
`72
`128
`
`0
`18
`52
`522
`0
`1490
`0
`26
`840
`13
`3890
`0
`2090
`598
`0
`81
`223
`441
`140
`1310
`0
`1120
`519
`0
`7590
`202
`0
`84
`233
`61
`118
`1560
`0
`8290
`21
`20
`70
`
`0
`14
`27
`150
`0
`603
`0
`21
`318
`0
`2230
`0
`959
`202
`0
`35
`134
`205
`56
`236
`0
`294
`78
`0
`755
`10
`0
`22
`47
`0
`9
`167
`0
`398
`21
`11
`24
`
`Time Point
`
`0 HR
`36 HR NB
`36 HR B
`0 HR
`24 HR NB
`24 HR B
`36 HR NB
`36 HR B
`0 HR
`24 HR NB
`
`18
`1
`26
`118
`0
`198
`2
`53
`299
`6
`12
`2
`353
`1
`1
`5
`108
`111
`22
`138
`4
`89
`15
`2
`4860
`4
`3
`34
`11
`1
`2
`91
`1
`67
`52
`22
`36
`
`37
`14
`19
`343
`0
`710
`0
`768
`2180
`0
`7070
`0
`2760
`3000
`0
`310
`250
`241
`41
`471
`0
`308
`108
`0
`2810
`10
`0
`26
`83
`0
`33
`96
`0
`4600
`124
`64
`87
`
`(B)
`
`126
`38
`69
`1490
`0
`2030
`1230
`1170
`4890
`13
`11800
`17
`7650
`3930
`22
`426
`992
`1320
`257
`1020
`5
`647
`320
`0
`3840
`78
`0
`116
`258
`0
`81
`86
`4
`13500
`1250
`528
`446
`
`34
`20
`15
`734
`0
`1090
`0
`1050
`3160
`11
`8960
`6
`3090
`2300
`9
`369
`493
`573
`116
`813
`4
`541
`130
`0
`3000
`33
`3
`63
`181
`2
`50
`26
`6
`4870
`107
`53
`89
`
`DIA-D
`
`200
`
`4690
`1160
`14700
`2120
`92
`2380
`131
`58
`15800
`672
`
`15
`1
`16
`141
`1
`202
`2
`105
`247
`5
`18
`2
`366
`2
`1
`4
`230
`66
`30
`1770
`1
`1110
`255
`1
`4490
`15
`2
`45
`123
`1
`18
`942
`1
`2490
`44
`21
`35
`
`15
`0
`21
`98
`0
`476
`1
`52
`387
`4
`12
`0
`516
`1
`2
`6
`125
`45
`24
`137
`1
`101
`24
`1
`3790
`5
`0
`16
`18
`0
`2
`65
`0
`183
`62
`27
`51
`
`DIA-E
`
`200
`
`3830
`752
`2460
`3340
`161
`3720
`324
`2770
`34600
`951
`
`4
`6
`15
`2
`17
`247
`8
`3
`2
`9
`4930
`2
`1720
`243
`0
`26
`44
`41
`ND
`605
`220
`576
`312
`0
`1540
`43
`0
`123
`315
`0
`46
`1380
`8
`6270
`0
`676
`0
`
`2
`9
`13
`1
`11
`150
`5
`1
`1
`6
`2450
`2
`859
`114
`0
`39
`47
`52
`ND
`1060
`3
`615
`453
`0
`1930
`79
`1
`141
`167
`1
`67
`1420
`4
`2420
`392
`192
`241
`
`1
`21
`14
`3
`8
`236
`12
`2
`1
`7
`1620
`2
`577
`103
`0
`19
`36
`79
`ND
`908
`2
`778
`357
`0
`1640
`123
`1
`121
`120
`2
`44
`890
`2
`1790
`728
`370
`663
`
`DIA-X
`
`200
`
`14000
`309
`7720
`5560
`505
`6230
`1010
`2750
`36100
`3600
`
`3
`0
`7
`9
`1
`12
`1
`6
`9
`0
`10
`0
`12
`9
`0
`7
`11
`11
`12
`12
`1
`12
`12
`0
`12
`5
`0
`9
`11
`1
`7
`11
`0
`12
`9
`8
`10
`
`DIA-Y
`
`200
`
`4040
`1490
`15000
`9
`140
`2890
`8
`11
`293
`571
`
`q 2005 Lippincott Williams & Wilkins
`
`(continued on next page)
`
`187
`
`Geneoscopy Exhibit 1022, Page 5
`
`

`

`Olson et al
`
`Diagn Mol Pathol  Volume 14, Number 3, September 2005
`
`TABLE 2. (continued ) Results of DNA Integrity Assay for DIA-positive Samples Incubated at Room Temperature for Prescribed
`Times, With and Without Stabilization Buffer. DIA Scores (A) and Total Recoverable DNA (B) are Shown for 11 Samples Found to
`be DIA-positive at t0
`
`(B)
`
`DIA-D
`
`DIA-E
`
`DIA-X
`
`DIA-Y
`
`Sample
`
`Time Point
`
`GP38
`
`GP31
`
`107
`
`117
`
`97
`
`105
`
`90
`
`GP79
`
`24 HR B
`36 HR NB
`36 HR B
`0 HR
`36 HR NB
`36 HR B
`0 HR
`36 HR NB
`36 HR B
`0HR
`48 HR NB
`48 HR B
`0HR
`48 HR NB
`48 HR B
`0HR
`48 HR NB
`48 HR B
`0HR
`48 HR NB
`48 HR B
`0HR
`48 HR NB
`48 HR B
`0 HR
`48 HR NB
`48 HR B
`
`200
`
`68200
`279
`29400
`35500
`103
`6320
`4100
`7220
`3400
`17900
`23
`3600
`6230
`4
`12100
`2790
`16
`1400
`1690
`24
`379
`36000
`175
`72900
`4470
`3680
`3980
`
`200
`
`72700
`471
`31600
`46400
`375
`10300
`3290
`6390
`1080
`6890
`21
`1860
`2620
`4
`7120
`2100
`12
`1180
`1420
`30
`549
`1070
`244
`30900
`20400
`14600
`14100
`
`200
`
`193
`517
`27400
`28
`284
`7510
`5960
`3060
`4990
`14900
`15
`3710
`3340
`3
`9020
`2260
`33
`1920
`1660
`13
`469
`15900
`54
`22700
`12200
`9790
`8510
`
`200
`
`61400
`77
`24000
`16700
`47
`7930
`2510
`3230
`ND
`5970
`23
`1790
`2830
`3
`3770
`2090
`22
`1690
`1320
`29
`626
`9520
`198
`10500
`13200
`6370
`8190
`
`( = DIA-positive marker; ( = DIA-positive sample.
`
`DNA component, and measures must be taken to minimize
`their negative impact. In this study, we investigate the in-
`fluence of adding EDTA-containing buffers to complex sam-
`ples, such as stool, to preserve DNA.
`One of the characterizing features of cancer is loss of
`normal cell regulation, including escape from apoptosis. It has
`been proposed that CRC may lead to enhanced exfoliation of
`colonocytes.20 Further, several groups 6,13,21,22 have reported
`an association of increased recovery of DNA from stools of
`patients found to have colorectal cancer. Similarly, detection of
`cell-free DNA in plasma and serum has received much atten-
`tion as a potential marker for detection and monitoring of
`cancers. There are many accounts of analysis of circulating
`DNA for cancer-related genetic mutations.23 Also, associa-
`tions of increased quantity24–27 and molecular size28,29 of cell-
`free DNA detected in plasma and serum with common cancers
`including CRC have been reported. The DIA assay was devel-
`oped to interrogate for the presence of long DNA,11 as an
`extension to the detection of DNA-quantity in bodily fluids, to
`increase assay sensitivity. It has also been shown that the DIA
`assay adds increased sensitivity to an assay based on a panel of
`mutations in known CRC-associated genes,6,8 indicating it is
`
`188
`
`an important marker. Our results indicate that without taking
`steps to preserve the integrity of DNA in stool samples, the
`majority of DIA-positive samples (ie, 82% [9/11] in this study)
`will be degraded leading to false-negative results. Although
`the duration that clinical samples may be subjected to un-
`favorable temperatures may vary in practice during sample
`transport, we believe that there is a need to develop a stan-
`dardized method for sample collection and handling to pre-
`serve this important marker. Addition of stabilization buffer to
`stool samples prior to transport presents an easily implemented
`solution that appears to be highly effective. We observed some
`variation in copy numbers of long DNA fragments comparing
`samples incubated with buffer versus samples analyzed fresh,
`but these differences were inconsequential with respect to DIA
`scores. All of the samples (11/11, 100%) that were DIA posi-
`tive at t0 remained positive after incubating at room tempera-
`ture for $36 hours when buffer was added to the stool sample,
`whereas without buffer addition only 18% of the samples
`(2/11) remained positive.
`A total of 43 samples were analyzed in this study. Thirty-
`eight were evaluated by the DIA assay, 11 of which were found
`to be DIA positive, as discussed earlier. Eleven samples were
`
`q 2005 Lippincott Williams & Wilkins
`
`Geneoscopy Exhibit 1022, Page 6
`
`

`

`Diagn Mol Pathol  Volume 14, Number 3, September 2005
`
`DNA Stabilization
`
`TABLE 3. Gene Mutation Analysis Results for Stool Aliquots Incubated At
`Room Temperature
`
`Sample
`
`Conditions
`
`DNA Quant.
`
`Marker
`
`SBE Threshold
`
`SBE Signal
`
`GP-003
`
`GP-023
`
`GP-024
`
`GP-025
`
`GP-026
`
`GP-034
`
`GP-029
`
`GP-030
`
`GP-031
`
`GP-105
`
`GP-121
`
`t0
`72 h
`t0
`72 h
`t0
`72 h
`t0
`48 h
`72 h
`t0
`48 h
`72 h
`t0
`48 h
`72 h
`t0
`36 h
`t0
`36 h
`36 h, Buffer
`t0
`36 h
`36 h, Buffer
`t0
`36 h
`36 h, Buffer
`t0
`48 h
`48 h, Buffer
`t0
`48 h
`48 h, Buffer
`
`2,180
`351
`
`59,800
`3,250
`509
`119
`217
`788
`121
`125
`458
`160
`185
`72,300
`1,180
`10,700
`1,110
`11,000
`501
`135
`338
`10,300
`13,500
`4,550
`1,540
`78
`9,140
`4,720
`1,500
`1,040
`
`Kras, k12p2
`
`p53, 273p1
`
`Kras, k12p2
`
`BAT-26
`
`0.400
`
`0.200
`
`0.400
`
`0.050
`
`p53, 248p2
`
`1.000
`
`p53, 175p2
`
`0.400
`
`p53, 248p2
`
`p53, 175p2
`
`1.000
`
`0.400
`
`Kras, k13p2
`
`0.400
`
`APC, 1309
`
`0.055
`
`APC, 1554
`
`0.250
`
`p53, 175p2
`
`0.400
`
`0.445
`1.504
`0.999
`1.705
`1.071
`2.423
`0.093
`0.380
`0.092
`6.648
`1.206
`3.697
`1.459
`1.914
`0.449
`1.505
`4.834
`1.229
`1.721
`1.793
`1.814
`3.650
`2.593
`0.219
`0.090
`0.210
`1.210
`0.049
`2.392
`2.699
`1.601
`2.329
`
`P/N
`
`POS
`POS
`POS
`POS
`POS
`POS
`POS
`POS
`POS
`POS
`POS
`POS
`POS
`POS
`POS
`POS
`POS
`POS
`POS
`POS
`POS
`POS
`POS
`POS
`POS
`POS
`POS
`NEG
`POS
`POS
`POS
`POS
`
`Sample handling conditions and analysis frequency is described in the materials and methods section. All aliquots were
`analyzed for total recoverable DNA (DNA Quant) as well as for the presence of any of 22 mutations in K-ras, APC, and p53 genes,
`as well as BAT-26.
`
`analyzed by the mutation panel assay of 22 point mutations.
`Six of the samples were analyzed by both DIA and the muta-
`tion panel. Analyzing all of the samples together (N = 43), sta-
`tistical analysis (1-tailed paired t test) shows a significant
`decrease (P = .0018) in recovered DNA for aliquots that had
`been incubated at room temperature without buffer (control = t0).
`The addition of buffer to samples prior to room temperature
`incubation was found to yield a significant increase in DNA
`recovery, relative to the matched aliquots with no buffer added
`(P = .010). Addition of stabilization buffer prior to room
`temperature incubation preserves DIA positives for all sample