SEARCH

SEARCH BY CITATION

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Authorship
  8. Acknowledgements
  9. References
  10. Supporting Information

Background

Current approaches to the detection of colorectal neoplasia associated with inflammatory bowel disease (IBD-CRN) are suboptimal.

Aim

To test the feasibility of using stool assay of exfoliated DNA markers to detect IBD-CRN.

Methods

This investigation comprised tissue and stool studies. In the tissue study, gene sequencing and methylation assays were performed on candidate genes using tissue DNA from 25 IBD-CRNs and from 25 IBD mucosae without CRN. Mutations on p53, APC, KRAS, BRAF or PIK3CA genes were insufficiently informative, but several aberrantly methylated genes were highly discriminant. In the stool study, we evaluated candidate methylated genes (vimentin, EYA4, BMP3, NDRG4) in a prospective blinded study on buffered stools from 19 cases with known IBD-CRN and 35 age- and sex-matched IBD controls without CRN. From stool-extracted DNA, target genes were assayed using quantitative allele-specific real-time target and signal amplification method.

Results

IBD-CRN cases included 17 with ulcerative colitis (UC) and two with Crohn's disease (CD); nine had cancer and 10 had dysplasia. Controls included 25 with UC and 10 with CD. Individually, BMP3, vimentin, EYA4 and NDRG4 markers showed high discrimination in stools with respective areas under the ROC curve of 0.91, 0.91, 0.85 and 0.84 for total IBD-CRN and of 0.97, 0.97, 0.95 and 0.85 for cancer. At 89% specificity, the combination of BMP3 and mNDRG4 detected 9/9 (100%) of CRC and 80% of dysplasia, 4/4 (100%) of high grade and 4/6 (67%) of low grade.

Conclusion

These findings demonstrate the feasibility of stool DNA testing for non-invasive detection of colorectal neoplasia associated with inflammatory bowel disease.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Authorship
  8. Acknowledgements
  9. References
  10. Supporting Information

Patients with inflammatory bowel disease (IBD) are at increased risk of colorectal neoplasia (CRN), including colorectal cancer (CRC).[1, 2] Factors known to increase CRC risk in IBD include duration and extent of chronic ulcerative colitis (CUC) or Crohn's colitis (CD), presence of primary sclerosing cholangitis (PSC), degree of histological activity and family history of CRC.[3-6] To reduce CRC risk, patients with IBD undergo periodic surveillance colonoscopies with multiple random biopsies to detect early visible and occult CRN (dysplasia and cancer).[7]

Limitations of this colonoscopic surveillance, as currently practiced, include under-sampling with random biopsies, an unknown ideal frequency for performing the surveillance exam and low grade of evidence for effectiveness.[8-10] Some centres use image-enhancing techniques, such as chromoendoscopy for surveillance. This has the advantage of identifying more dysplastic lesions by targeted rather than random biopsies,[11] but requires special training and sometimes extended endoscopy time. However, regardless of the surveillance technique used, CRN may be missed due to difficulty visualising neoplastic lesions, which are obscured against a background of chronic inflammatory changes.[12, 13] Identifying biomarkers that can provide complementary information to colonoscopy could fill an important clinical need in this patient population.

Stool assay of exfoliated molecular markers represents a non-invasive approach that could serve as such an adjunct to colonoscopy.[14, 15] While next-generation assay methods have yielded high detection rates for both sporadic CRC and precancer,[16-18] stool DNA testing as an approach to neoplasia detection in the IBD population has not been explored.

Tissue-based studies have demonstrated that IBD-CRN is associated with numerous molecular alterations, including acquired mutations in p53,[19, 20] APC,[21] K-ras[22-24] and BRAF[25] as well as aberrant methylation in EYA4,[26] ER, p16, MYOD, p14, E-cadherin, RUNX3, MINT1 and COX-2.[27-31] Several other genes, such as BMP3, vimentin (VIM),[32] septin 9[33] and NDRG4,[16] are selectively methylated in sporadic CRC, but have not been investigated in IBD.

The aims of this investigation were to (i) assess the discriminant value of the mutation markers p53, APC, BRAF, K-ras and PIK3CA and the methylation markers VIM, BMP3, EYA4 and septin 9 for detection of IBD-CRN based on DNA extracted from well-characterised tissue specimens and (ii) using the most discriminant tissue markers, prospectively assess the feasibility of stool DNA testing for the detection of premalignant and malignant IBD-CRN.

Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Authorship
  8. Acknowledgements
  9. References
  10. Supporting Information

Our investigation was approved by the Institutional Review Boards at: Mayo Clinic, Rochester, Minnesota, USA; University of Chicago, Chicago, Illinois, USA; and Mount Sinai School of Medicine, New York, New York, USA.

Tissue study

Patients. Tissues were identified from a single-centre archive of IBD-CRC case and IBD control specimens after confirmation of histological diagnosis. Cases and controls were matched for age (within a 10-year range), gender, disease duration, anatomic extent (left-sided/extensive) and PSC status (yes/no). DNA was extracted from paraffin-embedded tissues as described.[34]

Mutation Marker Gene Sequencing. Candidate exons on APC, p53, K-ras, BRAF and PIK3CA were amplified using real-time PCR (see Data S1).

Real-Time Methylation-Specific PCR (MSP). After bisulfite treatment, MSP was performed on VIM, BMP3 and septin 9 using Taq polymerase (Invitrogen, Carlsbad, CA, USA) and on EYA4 using SYBR Green master mix (Roche, Mannheim, Germany).

Stool study

Patients. Case patients with established IBD-CRN were recruited. Those who had undergone endoscopic or surgical treatment of neoplasia or with a history of other neoplasia of the gastrointestinal tract or respiratory system were excluded. Each site recruited IBD control patients undergoing surveillance colonoscopy, matched on age (in 5-year strata) and sex. As anticipated,[35] matching on more variables was not possible during prospective enrolment, but data was collected on IBD diagnosis (CD/UC/indeterminate), IBD duration, extent of colitis and PSC. IBD activity was assessed by a single expert pathologist, using a previously published protocol.[36] After informed consent, participants were given a container and toilet seat mounting bracket kit to collect stools prior to or at least 1 week after colonoscopy or sigmoidoscopy.[37, 38]

Sequence-specific gene capture. A 2-gramme equivalent of stool supernatant was used for multiplex capture of gene targets (β-actin, VIM, EYA4, BMP3 and NDRG4) by amino conjugated oligonucleotides complementary to target sequences (see Data S1).[39]

Assay of Methylated Markers. After capture, target DNA was bisulfite treated and quantitative allele-specific real-time target and signal amplification (QuARTS) reactions were performed on Roche 480 LightCyclers (Indianapolis, IN, USA), as described (see Data S1).[16] EYA4 methylation was assayed using methylation specific PCR, performed on a LightCycler 480 using SYBR Green I Master (Roche) as described.[39]

Statistical analysis

Based on a comparison of immunochemical faecal occult blood testing against colonoscopy for the detection of sporadic CRN,[40] the feasibility for IBD-CRN detection by stool DNA testing at this initial phase of evaluation was defined a priori as sensitivity for neoplasia >40%. Based on conservative prestudy assumptions, it was estimated that 15 patients in the case group would provide 80% power to distinguish a true sensitivity of 70% from a null value of 40% with a one-sided one-sample proportion test at the 5% level. The distributions of each marker as a continuous variable were compared between cases and controls using the Wilcoxon rank-sum test (JMP v8.0; SAS Institute, Cary NC, USA). Logistic regression was used to calculate receiver operating characteristics (ROC) curves, from which specificity cut-offs were imputed and marker sensitivities [with 95% confidence intervals (CI)] were calculated. To further study the effects of known prognostic factors on marker levels, differences in baseline variables between cases and controls were tested using Chi-squared for proportions and Wilcoxon rank-sum for continuous data. When baseline variables were significantly different, case and control marker results were stratified to assess confounding. Univariate and multivariate logistic regression models assessed potential interaction by age, sex and clinical risk factors, including comorbid PSC (yes/no), disease duration (in years) and disease extent (left-sided/extensive). anova was used to assess possible associations between IBD activity (inactive/mild/moderate/severe) and marker levels.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Authorship
  8. Acknowledgements
  9. References
  10. Supporting Information

Tissue study

Clinical characteristics were well-matched between cases and controls (Table 1). Figure 1 summarises the results of DNA sequencing for the case samples. Across six APC regions overlapping the mutation cluster region (1, 2, C, N, Y, L2), only three mutations were found. Four mutations were found on K-ras. As anticipated, p53 was the most informative marker with 11 mutations detected; however, these were spread out across a wide range of sites on all five exons tested. No mutations were identified on BRAF or PIK3CA. While specificity was 100% (no mutations found among control tissues), aggregate sensitivity using all 14 mutation markers combined was only 60%. This is similar to observed rates of DNA mutations assayed from tissues of sporadic CRC and advanced adenomas.[41]

Table 1. Patient characteristics for tissue study
  Cases N = 25 Controls N = 25
  1. CUC, chronic ulcerative colitis; PSC, primary sclerosing cholangitis; s.d., standard deviation.

  2. Cases = Colorectal cancer in CUC, Controls = CUC without neoplasia.

Male (%)16 (64)17 (68)
Mean age, years (s.d.)52 (14.4)50 (11.9)
Mean CUC duration, years (s.d.)20.7 (9.2)19.9 (8.3)
Extensive (%)21 (84)20 (80)
PSC (%)4 (16)3 (12)
image

Figure 1. Gene mutations detected in tissue DNA from inflammatory bowel disease associated cancers (n = 25).

Download figure to PowerPoint

For each of the methylation markers, ROC curves were constructed. Areas under the curve (AUC) were 0.97, 0.87, 0.81 and 0.73 for methylated EYA4 (mEYA4), VIM (mVIM), BMP3 (mBMP3) and Septin 9 respectively. Thus, mEYA4, mVIM and mBMP3 were selected for stool DNA testing. In addition, methylated NDGR4 (mNDRG4) was also selected because of its high discrimination for sporadic CRN in studies performed after the completion of the tissue study.[16]

Stool study

Given the high discrimination observed with methylation markers in the tissue study, an analysis of stool from independent sets of cases and controls was performed. Between 1 January 2009 and 31 October 2011, a total of 23 eligible cases and 220 eligible controls were identified and contacted. Nineteen IBD case patients with biopsy-confirmed CRN and 35 IBD control patients without CRN submitted stools (Table 2). Although the proportions of IBD diagnoses and comorbid PSC were not significantly different between the two groups, cases had significantly longer disease duration (= 0.0008) and were significantly more likely to have extensive disease involvement (P = 0.01). There was no difference in disease activity between cases and controls (P = 0.44).

Table 2. Patient characteristics for stool study
  Cases N = 19 Controls N = 35
  1. CUC, chronic ulcerative colitis; IBD, inflammatory bowel disease; PSC, primary sclerosing cholangitis.

  2. Cases = IBD with colorectal neoplasia, Controls = IBD without neoplasia.

  3. a

     = 0.0008

  4. b

     Inflammation proximal to splenic flexure.

  5. c

     P = 0.01

  6. d

     Disease activity could not be confirmed for three control patients.

CUC1725
Crohn's disease210
% Male6363
Median age, years (range)60 (45–72)60 (45–77)
Median IBD duration, years (range)30 (2–50)14 (0–45)a
Extensive (%)b17 (89)19 (54)c
IBD activityd
Inactive (%)8 (42)12 (38)
Mild (%)9 (45)11 (34)
Moderate (%)1 (5)3 (9)
Severe (%)1 (5)6 (19)
PSC (%)4 (21)5 (16)

Case neoplasms included nine cancers with a median size of 2.3 cm (range: 0.8–5 cm). Six of the nine (67%) were proximal to the splenic flexure. Median stage[42] was I (range I–IIIC). Additional neoplasms included eight discrete polypoid dysplastic lesions [three high-grade dysplasia (HGD), five low-grade dysplasia (LGD)] with a median size of 2.3 cm (range: 1.0–6.2) and two flat lesions (one HGD, one LGD) detected on random biopsy (size unknown).

β-actin, a marker of human DNA recovery, amplified in all case and control samples; therefore all patients were included in the analysis. All four markers individually showed high discrimination for cancer (Figure 2). AUCs with mBMP3, mVIM, mEYA4 and mNDRG4 were 0.97, 0.97, 0.95 and 0.85 respectively. For IBD-CRN the AUC with mBMP3, mVIM, mEYA4 and mNDRG4 were 0.91, 0.91, 0.85 and 0.84 respectively. For dysplasia, the AUC with mBMP3, mVIM, mEYA4 and mNDRG4 was 0.84, 0.85, 0.75 and 0.77 respectively. Stool assay of mBMP3 alone at 91% specificity was 100% (9/9) sensitive for CRC, 70% (7/10) of dysplasia and 84% (16/19) sensitive for all CRN (Table 3). At 89% specificity, the combination of mBMP3 and mNDRG4 detected 9/9 (100%) of CRC and 80% of dysplasia, 4/4 (100%) of high grade and 4/6 (67%) of low grade.

Table 3. IBD-associated colorectal neoplasm detection rates by stool assay of methylated DNA markers
Specificity cut-off, %Sensitivity, % (95% CI)
mBMP3 mVIM mEYA4 mNDRG4
  1. a

     CRC, colorectal cancer.

  2. b

     Neoplasia = CRC + premalignant dysplasia combined.

CRCa
9489 (51–99)89 (51–99)66 (31–91)44 (15–77)
91100 (63–100)89 (51–99)78 (40–96)44 (15–77)
89100 (63–100)89 (51–99)100 (63–100)100 (63–100)
Neoplasiab
9468 (43–86)68 (43–86)53 (29–74)37 (17–61)
9184 (60–96)68 (43–86)63 (39–82)37 (17–61)
8984 (60–96)68 (43–86)74 (48–90)74 (48–90)
Dysplasia
9450 (20–80)50 (20–80)40 (14–73)30 (8–65)
9170 (35–91)50 (20–80)50 (20–80)30 (8–65)
8970 (35–91)50 (20–80)50 (20–80)50 (20–80)
image

Figure 2. Receiver operating characteristics curve for detection of neoplasms by stool assay of methylated genes. Data plotted for (a) BMP3, (b) Vimentin, (c) EYA4 and (d) NDRG4 gene markers. AUC, area under curve; CRC, colorectal cancer.

Download figure to PowerPoint

The dynamic range of methylated copy numbers between cases and controls was wide for each stool marker (Figure 3). Among cases, copy numbers of mBMP3, mVIM, mEYA4 or mNDRG4 were not significantly different for proximal vs. distal neoplasms (P = 0.58, 0.73, 0.83 and 0.85, respectively). After stratifying for case vs. control status, marker levels were not significantly different when comparing patients with CUC and Crohn's disease.

image

Figure 3. Distributions of methylated gene marker levels in stools from IBD cases with colorectal neoplasia and from IBD controls without neoplasia. Data plotted for (a) BMP3, (b) Vimentin, (c) EYA4 and (d) NDRG4 gene markers. CRC, colorectal cancer; HGD, high-grade dysplasia; LGD, low-grade dysplasia.

Download figure to PowerPoint

In multivariate analyses, methylation markers for CRN detection remained significant in models which included age, sex, disease duration, disease extent or the presence of PSC (Table S1). ANOVA did not demonstrate any association between markers and disease activity for either cases or controls. Disease duration showed weak correlation with marker levels by univariate linear regression; however, when stratified by case and control status, this association was no longer significant for any of the four methylation markers evaluated. Furthermore, there was no association between anatomic extent of disease (extensive vs. left-sided) and marker levels for either cases or controls.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Authorship
  8. Acknowledgements
  9. References
  10. Supporting Information

Using DNA methylation markers, which were discriminant in tissue, we found that the stool assay achieved high detection rates of both CRC and dysplasia in IBD patients. For example, stool assay of a single informative marker, mBMP3, detected 100% of CRC and 84% of all neoplasms at 91% specificity. Importantly, stool marker levels assayed remained unaffected by neoplasm site within the colorectum, as we have observed with sporadic colorectal neoplasia.[16] These early results surpassed our predetermined threshold for feasibility.

While corroborative studies are clearly needed, our data suggest the potential usefulness of stool DNA testing to inform the frequency and rigour of colonoscopic surveillance. A non-invasive test, which could be performed without bowel cleansing in a patient's own home, might improve compliance with surveillance, which is currently poor, even among high-risk patients.[43, 44] Algorithms incorporating stool DNA as a complement to colonoscopy could potentially lengthen the interval between surveillance examinations in marker-negative patients, which could also reduce the high cost of surveillance endoscopy.[45] Conversely, a patient with a positive stool DNA test may benefit from colonoscopy at shorter surveillance intervals and/or using enhanced imaging techniques, such as chromoendoscopy.

The tissue study based on well-matched cases and controls showed that methylation markers are highly discriminant for IBD-CRN. These tissue findings corroborate our previous observations with mBMP3 and mEYA4.[26]

Prior studies of methylation markers in IBD-CRN have focused on tumour suppressor genes.[27, 29, 30, 46, 47] While BMP3 and NDRG4 are known tumour suppressors in CRC,[16, 48, 49] the biology need not be fully understood before a marker is clinically useful. The roles of EYA4 and VIM in IBD-carcinogenesis remain unclear, and these genes are aberrantly methylated in other tissues as well.[51, 50, 52] Studies have also demonstrated that mVIM is a sensitive stool marker for sporadic CRC.[54, 53]

Our study has several limitations. First, this was a case–control study sized to assess early feasibility of stool DNA testing for detection of IBD-CRN. While sample size was sufficient to meet the central aim, the power to evaluate subclasses among covariates and marker combinations was limited. Larger studies are needed to achieve greater precision for the sensitivity estimates. In addition, only two endoscopically inapparent (flat) dysplastic lesions were available for analysis, limiting inferences that can be drawn about discrimination for this endpoint. Nevertheless, the high sensitivity achieved by the combination of mBMP3 and mNDRG4 warrants further study, particularly when considering that these two markers have proved complementary for detection of sporadic CRN in studies with large sample sizes.[16-18] Methylated BMP3 detected more neoplasms in stool compared to tissue, which could reflect a high prevalence of sporadic-type CRN in IBD patients.[55] Second, while cases and controls in the stool study were well-matched on most variables, cases had a longer median duration of IBD and were more likely to have extensive disease. Accordingly, disease duration was further evaluated in stratified comparisons and multivariate models and was not found to significantly influence marker levels. Other parameters of disease severity, including anatomic extent of inflammation, degree of inflammation and presence of concomitant PSC, also had no effect on stool marker levels. Finally, conventional colonoscopy with nontargeted biopsies was used as the criterion standard, and this approach lacks sensitivity for CRN as currently practiced for IBD surveillance.[12, 13] Control patients who tested positive for methylation markers might therefore have had falsely negative colonoscopies, which would have affected specificity estimates. Underscoring the imperfect nature of colonoscopy, two of the nine CRC cases with positive stool results in this study were missed on colonoscopy and diagnosed only after colectomy.

These early results in tissue and stool represent an important first step in the evaluation of stool DNA as a non-invasive tool for detection of CRN in IBD patients. Additional studies are needed to corroborate and expand these novel findings. Particularly, prospective cohort studies conducted in the IBD surveillance setting will help determine how this non-invasive tool might improve colonoscopy yield and patient outcomes and potentially lower healthcare costs.

Authorship

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Authorship
  8. Acknowledgements
  9. References
  10. Supporting Information

Guarantor of the article: John B. Kisiel.

Author contributions: John B. Kisiel was involved in study design, data acquisition, analysis and drafting manuscript. Tracy C. Yab, Fareeda Taher Nazer Hussain and William R. Taylor were involved in data acquisition and analysis of data. Megan M. Garrity-Park contributed to the study design, data acquisition and analysis of data. William J. Sandborn, and Edward V. Loftus, Jr., were involved in critical revision of manuscript and conceptual input. Bruce G. Wolff was involved in the data acquisition, critical revision of manuscript and conceptual input. Thomas C. Smyrk was involved in the pathology interpretation, data acquisition and critical revision of manuscript. Steven H. Itzkowitz and David T. Rubin were involved in conceptual input, study supervision and manuscript review. Hongzhi Zou was involved in assay design and data analysis. Douglas W. Mahoney was involved in statistical analysis. David A. Ahlquist contributed to the study conceptual design, data analysis, drafting and editing of manuscript, obtained funding and study supervision. All authors approved the final version of the article, including the authorship list.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Authorship
  8. Acknowledgements
  9. References
  10. Supporting Information

Declaration of personal interests: Mayo Clinic is a minor equity investor in and has licenced intellectual property to Exact Sciences. Consistent with Mayo Clinic policy, Drs Kisiel and Ahlquist, Mr Taylor, and Ms Yab could share in potential future equity or royalties. Dr Itzkowitz is on the Scientific Advisory Board of Exact Sciences Corporation.

Declaration of funding interests: Funding was provided by a grant from the Charles Oswald Foundation. Dr Kisiel was supported by the Maxine and Jack Zarrow Family Foundation of Tulsa Oklahoma.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Authorship
  8. Acknowledgements
  9. References
  10. Supporting Information
  • 1
    Jess T, Loftus EV Jr, Velayos FS, et al. Risk of intestinal cancer in inflammatory bowel disease: a population-based study from olmsted county. Minnesota. Gastroenterology 2006; 130: 103946.
  • 2
    Howe HL, Wu X, Ries LA, et al. Annual report to the nation on the status of cancer, 1975–2003, featuring cancer among U.S. Hispanic/Latino populations. Cancer 2006; 107: 171142.
  • 3
    Itzkowitz SH, Harpaz N. Diagnosis and management of dysplasia in patients with inflammatory bowel diseases. Gastroenterology 2004; 126: 163448.
  • 4
    Cairns SR, Scholefield JH, Steele RJ, et al. Guidelines for colorectal cancer screening and surveillance in moderate and high risk groups (update from 2002). Gut 2010; 59: 66689.
  • 5
    Howdle P, Atkin M, Rutter M, et al. Colonoscopic surveillance for prevention of colorectal cancer in people with ulcerative colitis, Crohn's disease or adenomas. London: National Institute for Health and Clinical Excellence (UK), 2011.
  • 6
    Nuako KW, Ahlquist DA, Mahoney DW, Schaid DJ, Siems DM, Lindor NM. Familial predisposition for colorectal cancer in chronic ulcerative colitis: a case-control study. Gastroenterology 1998; 115: 107983.
  • 7
    Farraye FA, Odze RD, Eaden J, Itzkowitz SH. AGA technical review on the diagnosis and management of colorectal neoplasia in inflammatory bowel disease. Gastroenterology 2010; 138: 74674.
  • 8
    Karlen P, Kornfeld D, Brostrom O, Lofberg R, Persson PG, Ekbom A. Is colonoscopic surveillance reducing colorectal cancer mortality in ulcerative colitis? A population based case control study. Gut 1998; 42: 7114.
  • 9
    Loftus EV, Jr. Does monitoring prevent cancer in inflammatory bowel disease? J Clin Gastroenterol 2003; 36(Suppl.): S7983.
  • 10
    Collins PD, Mpofu C, Watson AJ, Rhodes JM. Strategies for detecting colon cancer and/or dysplasia in patients with inflammatory bowel disease. Cochrane Database Syst Rev 2006; 2: CD000279.
  • 11
    Subramanian V, Mannath J, Ragunath K, Hawkey CJ. Meta-analysis: the diagnostic yield of chromoendoscopy for detecting dysplasia in patients with colonic inflammatory bowel disease. Aliment Pharmacol Ther 2011; 33: 30412.
  • 12
    Connell WR, Lennard-Jones JE, Williams CB, Talbot IC, Price AB, Wilkinson KH. Factors affecting the outcome of endoscopic surveillance for cancer in ulcerative colitis. Gastroenterology 1994; 107: 93444.
  • 13
    Lim CH, Dixon MF, Vail A, Forman D, Lynch DA, Axon AT. Ten year follow up of ulcerative colitis patients with and without low grade dysplasia. Gut 2003; 52: 112732.
  • 14
    Imperiale TF, Ransohoff DF, Itzkowitz SH, Turnbull BA, Ross ME. Fecal DNA versus fecal occult blood for colorectal-cancer screening in an average-risk population. N Engl J Med 2004; 351: 270414.
  • 15
    Osborn NK, Ahlquist DA. Stool screening for colorectal cancer: molecular approaches. Gastroenterology 2005; 128: 192206.
  • 16
    Ahlquist DA, Zou H, Domanico M, et al. Next-generation stool DNA test accurately detects colorectal cancer and large adenomas. Gastroenterology 2012; 142: 24856.
  • 17
    Ahlquist DA, Taylor WR, Mahoney DW, et al. The stool DNA test is more accurate than the plasma septin 9 test in detecting colorectal neoplasia. Clin Gastroenterol Hepatol 2012;2727 e1.
  • 18
    Lidgard GP, Domanico M, Bruinsma JJ, et al. An optimized multi-marker stool test for colorectal cancer screening: initial clinical appraisal. Gastroenterology 2012; 142(Suppl. 1): S-770.
  • 19
    Taylor HW, Boyle M, Smith SC, Bustin S, Williams NS. Expression of p53 in colorectal cancer and dysplasia complicating ulcerative colitis. Br J Surg 1993; 80: 4424.
  • 20
    Lashner BA, Shapiro BD, Husain A, Goldblum JR. Evaluation of the usefulness of testing for p53 mutations in colorectal cancer surveillance for ulcerative colitis. Am J Gastroenterol 1999; 94: 45662.
  • 21
    Odze RD, Brown CA, Hartmann CJ, Noffsinger AE, Fogt F. Genetic alterations in chronic ulcerative colitis-associated adenoma-like DALMs are similar to non-colitic sporadic adenomas. Am J Surg Pathol 2000; 24: 120916.
  • 22
    Bell SM, Kelly SA, Hoyle JA, et al. c-Ki-ras gene mutations in dysplasia and carcinomas complicating ulcerative colitis. Br J Cancer 1991; 64: 1748.
  • 23
    Holzmann K, Klump B, Borchard F, et al. Comparative analysis of histology, DNA content, p53 and Ki-ras mutations in colectomy specimens with long-standing ulcerative colitis. Int J Cancer 1998; 76: 16.
  • 24
    Hirota Y, Tanaka S, Haruma K, et al. pS2 expression as a possible diagnostic marker of colorectal carcinoma in ulcerative colitis. Oncol Rep 2000; 7: 2339.
  • 25
    Aust DE, Haase M, Dobryden L, et al. Mutations of the BRAF gene in ulcerative colitis-related colorectal carcinoma. Int J Cancer 2005; 115: 6737.
  • 26
    Osborn NK, Zou H, Molina JR, et al. Aberrant methylation of the eyes absent 4 gene in ulcerative colitis-associated dysplasia. Clin Gastroenterol Hepatol 2006; 4: 2128.
  • 27
    Issa J-PJ, Ahuja N, Toyota M, Bronner MP, Brentnall TA. Accelerated age-related CpG island methylation in ulcerative colitis. Cancer Res 2001; 61: 35737.
  • 28
    Sato F, Shibata D, Harpaz N, et al. Aberrant methylation of the HPP1 gene in ulcerative colitis-associated colorectal carcinoma. Cancer Res 2002; 62: 68202.
  • 29
    Wheeler JM, Kim HC, Efstathiou JA, Ilyas M, Mortensen NJ, Bodmer WF. Hypermethylation of the promoter region of the E-cadherin gene (CDH1) in sporadic and ulcerative colitis associated colorectal cancer. Gut 2001; 48: 36771.
  • 30
    Garrity-Park MM, Loftus EV Jr, Sandborn WJ, Bryant SC, Smyrk TC. Methylation status of genes in non-neoplastic mucosa from patients with ulcerative colitis-associated colorectal cancer. Am J Gastroenterol 2010; 105: 16109.
  • 31
    Watanabe T, Kobunai T, Ikeuchi H, et al. RUNX3 copy number predicts the development of UC-associated colorectal cancer. Int J Oncol 2011; 38: 2017.
  • 32
    Zou H, Harrington JJ, Shire AM, et al. Highly methylated genes in colorectal neoplasia: implications for screening. Cancer Epidemiol Biomarkers Prev 2007; 16: 268696.
  • 33
    Grutzmann R, Molnar B, Pilarsky C, et al. Sensitive detection of colorectal cancer in peripheral blood by septin 9 DNA methylation assay. PLoS ONE 2008; 3: e3759.
  • 34
    Garrity-Park MM, Loftus EV Jr, Bryant SC, Sandborn WJ, Smyrk TC. Tumor necrosis factor-alpha polymorphisms in ulcerative colitis-associated colorectal cancer. Am J Gastroenterol 2008; 103: 40715.
    Direct Link:
  • 35
    Fletcher RH, Fletcher SW. Clinical Epidemiology. 4th edn. Philadelphia, PA: Lippincott Williams & Wilkins, 2005.
  • 36
    Gupta RB, Harpaz N, Itzkowitz S, et al. Histologic inflammation is a risk factor for progression to colorectal neoplasia in ulcerative colitis: a cohort study. Gastroenterology 2007; 133: 1099105;.
  • 37
    Zou H, Harrington JJ, Klatt KK, Ahlquist DA. A sensitive method to quantify human long DNA in stool: relevance to colorectal cancer screening. Cancer Epidemiol Biomarkers Prev 2006; 15: 11159.
  • 38
    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: 18391.
  • 39
    Kisiel JB, Yab TC, Taylor WR, et al. Stool DNA testing for the detection of pancreatic cancer: assessment of methylation marker candidates. Cancer 2012; 118: 262331.
  • 40
    Morikawa T, Kato J, Yamaji Y, Wada R, Mitsushima T, Shiratori Y. A comparison of the immunochemical fecal occult blood test and total colonoscopy in the asymptomatic population. Gastroenterology 2005; 129: 4228.
  • 41
    Ahlquist DA, Sargent DJ, Loprinzi CL, et al. Stool DNA and occult blood testing for screen detection of colorectal neoplasia. Ann Intern Med 2008; 149: 44150.
  • 42
    Edge SB, Byrd DR, Compton CC, Fritz AG, Greene FL, Trotti A (eds.). AJCC Cancer Staging Manual. 7th ed. New York: Springer, 2010.
  • 43
    Velayos FS, Liu L, Lewis JD, et al. Prevalence of colorectal cancer surveillance for ulcerative colitis in an integrated health care delivery system. Gastroenterology 2010; 139: 15118.
  • 44
    Vienne A, Simon T, Cosnes J, et al. Low prevalence of colonoscopic surveillance of inflammatory bowel disease patients with longstanding extensive colitis: a clinical practice survey nested in the CESAME cohort. Aliment Pharmacol Ther 2011; 34: 18895.
  • 45
    Rubenstein JH, Waljee AK, Jeter JM, Velayos FS, Ladabaum U, Higgins PD. Cost effectiveness of ulcerative colitis surveillance in the setting of 5-aminosalicylates. Am J Gastroenterol 2009; 104: 222232.
  • 46
    Sato F, Harpaz N, Shibata D, et al. Hypermethylation of the p14(ARF) gene in ulcerative colitis-associated colorectal carcinogenesis. Cancer Res 2002; 62: 114851.
  • 47
    Moriyama T, Matsumoto T, Nakamura S, et al. Hypermethylation of p14 (ARF) may be predictive of colitic cancer in patients with ulcerative colitis. Dis Colon Rectum 2007; 50: 138492.
  • 48
    Loh K, Chia JA, Greco S, et al. Bone morphogenic protein 3 inactivation is an early and frequent event in colorectal cancer development. Genes Chromosom Cancer 2008; 47: 44960.
  • 49
    Melotte V, Lentjes MH, van den Bosch SM, et al. N-Myc downstream-regulated gene 4 (NDRG4): a candidate tumor suppressor gene and potential biomarker for colorectal cancer. J Natl Cancer Inst 2009; 101: 91627.
  • 50
    Zou H, Osborn NK, Harrington JJ, et al. Frequent methylation of eyes absent 4 gene in Barrett's esophagus and esophageal adenocarcinoma. Cancer Epidemiol Biomarkers Prev 2005; 14: 8304.
  • 51
    Yang WD, Yab TC, Taylor WR, et al. Aberrant gene methylation in the neoplastic progression of Barrett's esophagus: identification of candidate diagnostic markers. Gastroenterology 2011; 5(Suppl. 1): S-222.
  • 52
    Moinova H, Leidner RS, Ravi L, et al. Aberrant vimentin methylation is characteristic of upper gastrointestinal pathologies. Cancer Epidemiol Biomarkers Prev 2012; 21: 594600.
  • 53
    Itzkowitz SH, Jandorf L, Brand R, et al. Improved fecal DNA test for colorectal cancer screening. Clin Gastroenterol Hepatol 2007; 5: 1117.
  • 54
    Itzkowitz S, Brand R, Jandorf L, et al. A simplified, noninvasive stool DNA test for colorectal cancer detection. Am J Gastroenterol 2008; 103: 286270.
    Direct Link:
  • 55
    Odze RD, Farraye FA, Hecht JL, Hornick JL. Long-term follow-up after polypectomy treatment for adenoma-like dysplastic lesions in ulcerative colitis. Clin Gastroenterol Hepatol 2004; 2: 53441.

Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Authorship
  8. Acknowledgements
  9. References
  10. Supporting Information
FilenameFormatSizeDescription
apt12218-sup-0001-TableS1-DataS1.docxWord document34K

Table S1. Results of multivariate models of association between clinical endpoints and methylation markers assayed from stool, adjusting for clinical variables.

Data S1. Supplemental methods.

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.