A Sensitive Method to Quantify Human Long DNA in Stool:
`Relevance to Colorectal Cancer Screening
`
`1115
`
`Hongzhi Zou, Jonathan J. Harrington, Kristie K. Klatt, and David A. Ahlquist
`
`Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, Minnesota
`
`Abstract
`
`Downloaded from http://aacrjournals.org/cebp/article-pdf/15/6/1115/2265348/1115.pdf by guest on 05 May 2023
`
`Human long DNA in stool may reflect nonapoptotic exfoli-
`ation and has been used as a colorectal cancer (CRC) marker.
`Targeting human-specific Alu repeats represents a logical but
`untested approach. A real-time Alu PCR assay was developed
`for quantifying long human DNA in stool and evaluated in
`this study. The accuracy and reproducibility of this assay and
`the stability of long DNA during room temperature fecal
`storage were assessed using selected patient stools and stools
`added to human DNA. Thereafter, long DNA levels were
`determined in blinded fashion from 18 CRC patients and 20
`colonoscopically normal controls. Reproducibility of real-time
`Alu PCR for quantifying fecal long DNA was high (r 2 = 0.99;
`P < 0.01). Long DNA levels in nonbuffered stools stored at
`
`room temperature fell a median of 75% by 1 day and 81% by 3
`days. An EDTA buffer preserved DNA integrity during such
`storage. Human long DNA was quantifiable in all stools but
`was significantly higher in stools from CRC patients than
`from normal controls (P < 0.05). At a specificity of 100%, the
`sensitivity of long DNA for CRC was 44%. Results indicate
`that real-time Alu PCR is a simple method to sensitively
`quantify long human DNA in stool. This study shows that not
`all CRCs are associated with increased fecal levels of long
`DNA. Long DNA degrades with fecal storage, and measures
`to stabilize this analyte must be considered for optimal
`(Cancer Epidemiol Biomarkers Prev
`use of this marker.
`2006;15(6):1115–9)
`
`Introduction
`
`Colorectal cancer (CRC) is the second leading cause of cancer-
`related death in the United States (1). Although CRC mortality
`is preventable if neoplasms can be detected at curable stage (2),
`only a minority of the population undergoes regular screening
`(3). Except
`for fecal occult blood testing, screening tools
`endorsed by the American Cancer Society are invasive and
`expensive (4).
`testing provides a
`The emergence of molecular stool
`possible user-friendly alternative to conventional methods of
`CRC screening. A variety of DNA markers have been detected
`in the stools (5), including mutations of oncogenes (6) and
`tumor suppressor genes (7), microsatellite instability (8), and
`DNA methylation (9, 10). Owing to the continuous exfoliation
`of nonapoptotic neoplastic cells,
`long DNA occurs more
`abundantly in CRC stools than normal ones and serves as a
`candidate screening marker (11, 12). Colonocytes shed from
`normal epithelium undergo apoptosis, and their DNA is
`broken down by endonucleases into fragments shorter than
`200 bp (12). However, there seems to be an escape from
`such apoptosis in exfoliated dysplastic cells, which results in
`long DNA sequences in stool that can be used for cancer
`detection (12).
`Present methods for detecting long DNA use assay of
`multiple-specific target sequences on different genes (12, 13).
`Assay of Alu sequences represents a potentially simple
`approach to measure human long DNA in stool. Alu
`sequences embody the largest family of middle repetitive
`DNA sequences in the human genome. An estimated half
`million Alu copies are present per haploid human genome
`(14). Because Alu sequences are so abundantly distributed
`throughout
`the genome and specific to the genomes of
`
`Received 12/28/05; revised 3/3/06; accepted 4/11/06.
`Grant support: Charles Oswald Foundation.
`The costs of publication of this article were defrayed in part by the payment of page charges.
`This article must therefore be hereby marked advertisement in accordance with 18 U.S.C.
`Section 1734 solely to indicate this fact.
`Requests for reprints: David A. Ahlquist, Division of Gastroenterology and Hepatology,
`Mayo Clinic, Rochester, MN 55905. Phone: 507-266-4338; Fax: 507-266-0350.
`E-mail: ahlquist.david@mayo.edu
`Copyright D 2006 American Association for Cancer Research.
`doi:10.1158/1055-9965.EPI-05-0992
`
`primates (14), an assay that amplifies DNA sequences longer
`than 200 bp within these 300-bp repeats should provide a
`genome-wide approach to quantify human long DNA in stool.
`Alu-based assays have been used to quantify human tumor
`xenograft burden in murine (15) or chicken embryo models
`(16) as well as integrated HIV-1 DNA in infected HeLa cells
`(17) but have not been applied to stool.
`This study was designed to (a) validate a real-time Alu PCR
`assay for quantifying human long DNA in stool, (b) evaluate
`the stability of long DNA in stool stored at room temperature
`and the effectiveness of an EDTA buffer for stabilizing DNA
`integrity, and (c) explore the feasibility of fecal long DNA
`quantification for CRC screening.
`
`Materials and Methods
`
`The study was approved by the Mayo Clinic Institutional
`Review Board.
`
`Stool DNA Extraction. Total DNA was extracted from stool
`samples with QIAamp DNA Stool Mini kit (Qiagen, Valencia,
`CA). Stool (2 g) was homogenized in 20 mL buffer ASL, and
`stool slurry (2 mL) was then used to extract total DNA
`following the instruction of
`the manufacturer. DNA was
`finally eluted in 100 AL buffer AE.
`Real-time Alu PCR. The Alu sequence consists of
`conserved regions and variable regions.
`In the putative
`consensus Alu sequence, the conserved regions are the 25-bp
`span between nucleotide positions 23 and 47 and 16-bp span
`between nucleotide positions 245 and 260 (14). Although
`primers may be designed in any part of the Alu sequences for
`more effectively amplifying Alu sequences, the PCR primers
`should completely or partially (at least the 3¶-regions of the
`primers) locate in the conserved regions. Primers specific for
`the human Alu sequences [sense (5¶-ACGCCTGTAATCC-
`CAGCACTT-3¶) and antisense (5¶-TCGCCCAGGCTG-
`GAGTGCA-3¶)] were used to amplify sequences f245 bp
`inside Alu repeats (Fig. 1; ref. 16). Stool DNA was diluted 1:5
`with 1 Tris-EDTA buffer (pH 7.5) for PCR amplification. Tris-
`EDTA buffer – diluted stool DNA (1 AL) was amplified in a
`total volume of 25 AL containing 1 iQ SYBR Green Supermix
`
`Cancer Epidemiol Biomarkers Prev 2006;15(6). June 2006
`
`Geneoscopy Exhibit 1052, Page 1
`
`

`

`1116 Quantification of Human DNA in Stool
`
`Figure 1. The design of the real-time Alu PCR. Primers with 3¶-ends complementary to the conserved regions of consensus sequence were used
`to amplify products f245 bp inside Alu repeats.
`
`Downloaded from http://aacrjournals.org/cebp/article-pdf/15/6/1115/2265348/1115.pdf by guest on 05 May 2023
`
`demographic and clinical characteristics of the CRC patients
`and controls are shown in Table 1. All stools were collected
`before colonoscopy or surgery. None of the CRC patients had
`undergone chemotherapy or radiotherapy before stool collec-
`tion. Any previous instrumentation had occurred >2 weeks
`before stool collection. A plastic bucket device was used to
`collect whole stool. Stools in sealed buckets were immediately
`transported to our laboratory, and total DNA was extracted
`from all stools within 4 hours from defecation.
`
`Statistical Analysis. For human long DNA levels obtained
`by real-time Alu PCR, the median for each group of stool
`samples was calculated, and Wilcoxon signed-rank test was
`used to compare the human long DNA levels of different stool
`groups. Spearman’s rank correlation was used to calculate the
`correlation coefficient of the reproducibility. Statistical tests
`were done using SAS statistical software (SAS Institute, Inc.,
`Cary, NC). All Ps were two sided.
`
`Results
`
`Validating Real-time Alu PCR Assay. To determine the
`dynamic range of the real-time Alu PCR, human genomic
`
`(Bio-Rad, Hercules, CA), 200 nmol/L each primer under the
`following conditions: 95jC for 3 minutes followed by 23 cycles
`of 95jC and 60jC for 30 seconds and 72jC for 40 seconds.
`Standard curve was created for each plate by amplifying 10-
`fold serially diluted human genomic DNA samples (Novagen,
`Madison, WI). Melting curve was made after each PCR to
`guarantee that only one product was amplified for all samples.
`Amplification was carried out in 96-well plates in an iCycler
`(Bio-Rad). Each plate consisted of stool DNA samples and
`multiple positive and negative controls. Each assay was done
`in duplicate.
`
`Long DNA Stability Analysis. Five fresh stools from CRC
`patients were used to test the stability of human long DNA in
`stool stored at room temperature. Four aliquots (2 g each) from
`each of the five stools were stored at room temperature for 0, 1,
`3, and 8 days. Total stool DNA was extracted from each aliquot
`with QIAamp DNA Stool Mini kit as described above. Human
`long DNA in total stool DNA sample was quantified with real-
`time Alu PCR as described above. Long DNA levels in stool
`aliquots extracted in days 1, 3, and 8 were divided by long
`DNA level in the stool aliquot extracted in day 0 for each stool
`sample to calculate the percentage of intact long DNA kept in
`the stool aliquots stored at room temperature for different
`durations. The median percentage of long DNA kept at each
`time point for five stools was then calculated.
`
`Stabilizing Human DNA Integrity. Four fresh normal
`stools with added human genomic DNA were used to test
`the effectiveness of an EDTA-based buffer for stabilizing DNA
`integrity in stools. Human genomic DNA (1 Ag) was spiked
`into two aliquots (4 g each) of each stool, and then aliquots of
`each stool were homogenized with 40 mL of two different
`buffers, including buffer with 100 mmol/L EDTA [0.5 mol/L
`Tris, 10 mmol/L NaCl, 100 mmol/L EDTA (pH 7); ref. 18] and
`buffer with 16 mmol/L EDTA [0.5 mol/L Tris, 10 mmol/L
`NaCl, 16 mmol/L EDTA (pH 7)]. Homogenized stool slurry
`was stored at room temperature, and 2 mL of it was used for
`stool DNA extraction at each of four different time points (day
`0, 1, 3, and 8). Total stool DNA was extracted from each aliquot
`with QIAamp DNA Stool Mini kit with some modifications.
`Human DNA in total stool DNA sample was quantified with
`real-time Alu PCR. The median percentage of human DNA
`kept at each time point was calculated.
`
`Clinical Pilot Study. A completely independent set of fresh
`stools from 18 CRC patients and from 20 colonoscopically
`normal individuals were analyzed in blinded fashion. The
`
`Table 1. Demographic and clinical characteristics of
`subjects
`
`Cancer
`
`Normal
`
`Number
`Sex (M/F)
`Mean age (y)
`Site (proximal/distal)
`Median size, cm (range)
`Stage (Dukes AB/CD)
`
`18
`12/6
`62
`5/13
`3.5 (1.1-10.0)
`8/9*
`
`20
`11/9
`71
`
`*Duke stage information was not available for a patient who did not have
`surgery.
`
`Figure 2. A. Human genomic DNA samples, which had been serially
`diluted by 10-fold (lines 1, 2.5 ng; lines 2, 250 pg; lines 3, 25 pg;
`lines 4, 2.5 pg; and lines 5, 250 fg), were amplified with real-time
`Alu PCR. Water control, 2.5 ng of each genomic DNA from pig,
`bovine, and chicken, and 2.5 ng E. coli genomic DNA were not
`amplified with real-time Alu PCR (lines 6). B. A standard curve was
`created with the log starting quantity and threshold cycle of the
`10-fold serially diluted human genomic DNA samples.
`
`Cancer Epidemiol Biomarkers Prev 2006;15(6). June 2006
`
`Geneoscopy Exhibit 1052, Page 2
`
`

`

`Downloaded from http://aacrjournals.org/cebp/article-pdf/15/6/1115/2265348/1115.pdf by guest on 05 May 2023
`
`Cancer Epidemiology, Biomarkers & Prevention 1117
`
`and Escherichia coli, a common bacterium in stool, were tested
`by this method. The Alu-based PCR assay was negative for all
`nonhuman mammalian DNA and E. coli DNA (Fig. 2A).
`Because stool contains PCR inhibitors (19), quantification
`could be affected by PCR inhibitors. To check whether assay
`accuracy was affected by potential PCR inhibitors, 500 pg
`human genomic DNA (2 AL) was added into 10 different
`stool DNA samples (38 AL each), and mixed DNA (1 AL),
`which contained 25 pg human genomic DNA, was then
`quantified with real-time Alu PCR. The mean recovery
`percentage of the added samples was 99.6% (range, 91.4-
`107.8%; Fig. 3A). For further confirming that PCR inhibitors
`did not affect the quantitative accuracy of the assay, one stool
`DNA sample from a CRC patient was 10-fold serially diluted
`and then quantified with real-time Alu PCR. Linear recovery
`of long DNA from these serially diluted stool DNA aliquots
`(r 2 = 0.997) confirmed the absence of interference by PCR
`inhibitors (Fig. 3B).
`The reproducibility of the real-time Alu PCR was studied in
`frozen stool samples from eight CRC patients and eight normal
`individuals. Human long DNA in these stool DNA samples
`was quantified in duplicate. The human long DNA levels of
`duplicate runs correlated highly (r 2 = 0.99; P < 0.01; Fig. 4).
`
`Instability of Human Long DNA in Stools Stored at Room
`Temperature. Compared with stools tested on day 0, median
`long DNA levels in stools stored at room temperature for 1, 3,
`and 8 days after defecation fell 75%, 81%, and 89%,
`respectively (Fig. 5A).
`From four fresh normal stools added to human DNA and
`mixed with low concentration EDTA (16 mmol/L), recoveries
`of human DNA after room temperature storage for 1, 3, and
`8 days were 65%, 19%, and 3%, respectively, compared with
`day 0. However, for stool aliquots mixed in buffer with high
`EDTA concentration (100 mmol/L), median recoveries of
`added human DNA were preserved at 121%, 118%, and
`100%, respectively (Fig. 5B).
`
`Human Long DNA Levels in CRC Stools and Normal
`Controls. Human long DNA levels in 18 CRC and 20 normal
`fresh stools, which were collected immediately after defeca-
`tion, were quantified by real-time Alu PCR in blinded
`fashion. Human long DNA was detected in all 38 stool
`samples but was significantly higher in CRC stools (median,
`309 ng/g stool; range, 5-21,115)
`than in normal stools
`(median, 70 ng/g stool; range, 2-2,870; P = 0.04; Fig. 6). At
`a long DNA cutoff of 2,900 ng/g stool, sensitivity for CRC
`was 44% (8/18), and specificity was 100% (20/20). Median
`long DNA in five proximal CRC stools was 48 ng/g (range,
`10-506 ng/g) and in 13 distal CRC stools was 4264 ng/g
`
`Figure 3. A. Human genomic DNA (25 pg) added into 10 different
`stool DNA samples was recovered by real-time Alu PCR. Recovery
`percentage (%) equals to human DNA amount recovered divided by
`human DNA amount added and then multiplied by 100. B. A stool
`DNA sample was 10-fold serially diluted, and human long DNA was
`then quantified using real-time Alu PCR. Linear recovery of human
`long DNA in serially diluted stool DNA samples.
`
`DNA samples serially diluted over a 10-fold range (2.5 ng,
`250 pg, 25 pg, 2.5 pg, 250 fg, and 25 fg) were amplified with
`the real-time Alu PCR. Alu sequences were linearly detected
`from 250 fg up to 2.5 ng human genomic DNA per PCR
`(Fig. 2A and B).
`To confirm the human specificity of the real-time Alu PCR,
`genomic DNA samples from pig, bovine, and chicken, which
`are three nonhuman species typically consumed in the diet,
`
`Figure 4. Stool DNA samples from eight CRC and
`eight normal stools were quantified with real-time Alu
`PCR twice. The human long DNA levels from these
`two runs showed good reproducibility (r 2 = 0.99).
`
`Cancer Epidemiol Biomarkers Prev 2006;15(6). June 2006
`
`Geneoscopy Exhibit 1052, Page 3
`
`

`

`Downloaded from http://aacrjournals.org/cebp/article-pdf/15/6/1115/2265348/1115.pdf by guest on 05 May 2023
`
`1118 Quantification of Human DNA in Stool
`
`(range, 5-21,115; P = 0.09). In this small series, tumor size did
`not significantly affect long DNA levels in stool.
`
`Discussion
`
`This report describes a new method to quantify human long
`DNA in stool using real-time PCR amplification of a 245-bp
`sequence within Alu repeats. The method is very sensitive
`with a dynamic range of 250 fg to 2.5 ng human genomic DNA,
`accurately detects human DNA added into stools, and yields
`highly reproducible results. Furthermore, this real-time Alu
`PCR method may have advantages of simplicity and speed
`compared with other approaches that describe use of multiple
`gene targets to assay long human DNA in stool (12, 13).
`With this validated new method, we found that human long
`DNA was present
`in all stools tested, but
`levels were
`significantly higher in stools from CRC patients than from
`normal individuals. When human long DNA in stool was
`used as a marker at a 100% specificity cutoff, about half of
`CRC patients could be detected, which is consistent with
`the performance of long DNA as a marker in earlier reports
`(11-13). The abundance of human long DNA in stools from
`CRC patients likely reflects the nonapoptotic exfoliation that
`occurs with CRC described by others (11-13).
`In two recent multicenter studies (20, 21), human long DNA
`in stool was less informative than in earlier reports. This
`discrepancy seems to be due to degradation by bacterial
`DNAases during prolonged preassay fecal storage that
`occurred with mailed-in samples in these studies. Experimen-
`tal observations in the present study and by others (18)
`corroborate the instability of human long DNA during fecal
`storage. Such degradation can be prevented by mixing stools
`with buffers containing a DNAase inhibitor like EDTA (18) as
`was shown in the present study. If human long DNA is to be
`
`Figure 6. Human long DNA levels of stools in the blinded pilot
`o
`clinical study.
`, stool sample. Solid horizontal bar, median of human
`long DNA concentration within a group of subjects.
`
`used clinically as a fecal marker, then attention must be given
`to incorporating a DNAase inhibitor as part of specimen
`collection and processing.
`Human long DNA is not specific for CRC. Preliminary
`reports suggest that human long DNA in stool may detect
`cancers in the upper gastrointestinal
`track as well
`(22).
`Inflammatory bowel disease has also been shown to be
`associated with elevated levels of human long DNA in stools
`(23). In contrast to normal epithelial cells, which undergo
`apoptosis (anoikis) when shed from their basement membrane
`attachment (24), inflammatory cells are anchorage indepen-
`dent and logically contribute to long DNA in stools. The
`discriminant value of human long DNA measured by this
`method would need to be verified in a larger and more
`representative sample if it were to be considered for screening
`or other clinical applications.
`Real-time Alu PCR is a simple, rapid, and inexpensive
`method for quantifying human long DNA in stools. This
`method may have useful applications for research observa-
`tions and clinical testing.
`
`Acknowledgments
`We thank Tammy S. Neseth and Ann Kolb for collecting samples.
`
`3.
`
`References
`Jemal A, Murray T, Ward E, et al. Cancer statistics 2005. CA Cancer J Clin
`1.
`2005;55:10 – 30.
`2. Mandel JS, Bond JH, Church TR, et al. Reducing mortality from colorectal
`cancer by screening for fecal occult blood. Minnesota Colon Cancer Control
`Study. N Engl J Med 1993;328:1365 – 71.
`From the centers for disease control and prevention. Colorectal cancer test
`use among persons aged > or = 50 years—United States, 2001. JAMA 2003;
`289:2492 – 3.
`4. Levin B, Brooks D, Smith RA, Stone A. Emerging technologies in screening
`for colorectal cancer: CT colonography, immunochemical fecal occult blood
`tests, and stool screening using molecular markers. CA Cancer J Clin 2003;
`53:44 – 55.
`5. Osborn NK, Ahlquist DA. Stool screening for colorectal cancer: molecular
`approaches. Gastroenterology 2005;128:192 – 206.
`Sidransky D, Tokino T, Hamilton SR, et al. Identification of ras oncogene
`mutations in the stool of patients with curable colorectal tumors. Science
`1992;256:102 – 5.
`7. Traverso G, Shuber A, Levin B, et al. Detection of APC mutations in fecal
`DNA from patients with colorectal tumors. N Engl J Med 2002;346:311 – 20.
`8. Traverso G, Shuber A, Olsson L, et al. Detection of proximal colorectal
`cancers through analysis of faecal DNA. Lancet 2002;359:403 – 4.
`9. Ahlquist DA, Klatt KK, Harrington JJ, Cunningham JM. Novel use of
`hypermethylated DNA markers in stool for detection of colorectal cancer: a
`feasibility study. Gastroenterology 2002;122 Suppl:A40.
`10. Muller HM, Oberwalder M, Fiegl H, et al. Methylation changes in faecal
`DNA: a marker for colorectal cancer screening? Lancet 2004;363:1283 – 5.
`11. Ahlquist DA, Skoletsky JE, Boynton KA, et al. Colorectal cancer screening by
`detection of altered human DNA in stool: feasibility of a multitarget assay
`panel. Gastroenterology 2000;119:1219 – 27.
`
`6.
`
`Figure 5. A. Long DNA levels of CRC stools stored at room
`temperature for 1, 3, and 8 days after defecation fell median
`percentages of 75%, 81%, and 89%, respectively. B. Buffers with
`100 mmol/L EDTA and 16 mmol/L EDTA showed different effects on
`preserving human DNA added in stools stored at room temperature.
`
`Cancer Epidemiol Biomarkers Prev 2006;15(6). June 2006
`
`Geneoscopy Exhibit 1052, Page 4
`
`

`

`Cancer Epidemiology, Biomarkers & Prevention 1119
`
`12. Boynton KA, Summerhayes IC, Ahlquist DA, Shuber AP. DNA integrity as a
`potential marker for stool-based detection of colorectal cancer. Clin Chem
`2003;49:1058 – 65.
`13. Calistri D, Rengucci C, Bocchini R, Saragoni L, Zoli W, Amadori D. Fecal
`multiple molecular tests to detect colorectal cancer in stool. Clin Gastro-
`enterol Hepatol 2003;1:377 – 83.
`14. Kariya Y, Kato K, Hayashizaki Y, Himeno S, Tarui S, Matsubara K.
`Revision of consensus sequence of human Alu repeats—a review. Gene
`1987;53:1 – 10.
`15. Schneider T, Osl F, Friess T, Stockinger H, Scheuer WV. Quantification of
`human Alu sequences by real-time PCR—an improved method to measure
`therapeutic efficacy of anti-metastatic drugs in human xenotransplants. Clin
`Exp Metastasis 2002;19:571 – 82.
`16. Zijlstra A, Mellor R, Panzarella G, et al. A quantitative analysis of rate-
`limiting steps in the metastatic cascade using human-specific real-time
`polymerase chain reaction. Cancer Res 2002;62:7083 – 92.
`17. Brussel A, Sonigo P. Analysis of early human immunodeficiency virus type
`1 DNA synthesis by use of a new sensitive assay for quantifying integrated
`provirus. J Virol 2003;77:10119 – 24.
`
`20.
`
`18. Olson J, Whitney DH, Durkee K, Shuber AP. DNA stabilization is critical for
`maximizing performance of fecal DNA-based colorectal cancer tests. Diagn
`Mol Pathol 2005;14:183 – 91.
`19. Deuter R, Pietsch S, Hertel S, Muller O. A method for preparation of fecal
`DNA suitable for PCR. Nucleic Acids Res 1995;23:3800 – 1.
`Imperiale TF, Ransohoff DF, Itzkowitz SH, Turnbull BA, Ross ME. Colorectal
`Cancer Study Group. Fecal DNA versus fecal occult blood for colorectal-cancer
`screening in an average-risk population. N Engl J Med 2004;351:2704 – 14.
`21. Ahlquist DA, Sargent DJ, Levin TR, et al. Stool DNA screening for colorectal
`cancer: prospective multicenter comparison with hemoccult. Gastroenterol-
`ogy 2005;128:63.
`J, Pearson R. Universal detection of
`Jett
`22. Ahlquist D, Cameron A,
`aerodigestive cancers by assay of nonapoptotic human DNA in stool.
`Gastroenterology 2002;118 Suppl:A855.
`Itzkowitz S, Jandorf L, Ullman T, et al. Stool DNA testing identifies patients
`with inflammatory bowel disease and dysplasia. Gastroenterology 2005;128:
`A567.
`24. Shanmugathasan M, Jothy S. Apoptosis, anoikis, and their relevance to the
`pathobiology of colon cancer. Pathol Int 2000;50:273 – 9.
`
`23.
`
`Downloaded from http://aacrjournals.org/cebp/article-pdf/15/6/1115/2265348/1115.pdf by guest on 05 May 2023
`
`Cancer Epidemiol Biomarkers Prev 2006;15(6). June 2006
`
`Geneoscopy Exhibit 1052, Page 5
`
`