FUTURE DIRECTIONS
AND
METHODS
FOR
IBD RESEARCH
Blood-based Biomarkers Used to Predict Disease Activity in Crohn’s Disease and Ulcerative Colitis Robert Burakoff, MD, MPH,* Vikas Pabby, MD, MPH,† Louisa Onyewadume, BA,* Robert Odze, MD,‡ Cheryl Adackapara, MD,‡ Wei Wang, PhD,* Sonia Friedman, MD,* Matthew Hamilton, MD,* Joshua Korzenik, MD,* Jonathan Levine, MD,* Frederick Makrauer, MD,* Changming Cheng, PhD,§ Hai Choo Smith, PhD,k Choong-Chin Liew, PhD,§,k and Samuel Chao, MASC¶
Background: Identifying specific genes that are differentially expressed during inflammatory bowel disease flares may help stratify disease activity. The aim of this study was to identify panels of genes to be able to distinguish disease activity in Crohn’s disease (CD) and ulcerative colitis (UC).
Methods: Patients were grouped into categories based on disease and severity determined by histological grading. Whole blood was collected by PAXgene Blood RNA collection tubes, (PreAnalytiX) and gene expression analysis using messenger RNA was conducted. Logistic regression was performed on multiple combinations of common probe sets, and data were evaluated in terms of discrimination by computing the area under the receiving operator characteristic curve (ROC–AUC). Results: Nine inactive CD, 8 mild CD, 10 moderate-to-severe CD, 9 inactive UC, 8 mild UC, 10 moderate-to-severe UC, and 120 controls were hybridized to Affymetrix U133 Plus 2 microarrays. Panels of 6 individual genes discriminated the stages of disease activity: CD with mild severity {ROC–AUC, 0.89 (95% confidence interval [CI], 0.84%–0.95%)}, CD with moderate-to-severe severity (ROC–AUC 0.98 [95% CI, 0.97–1.0]), UC with mild severity (ROC–AUC 0.92 [95% CI, 0.87–0.96]), and UC with moderate-to-severe severity (ROC–AUC 0.99 [95% CI, 0.97–1.0]). Validation by real-time reverse transcription–PCR confirmed the Affymetrix microarray data.
Conclusions: The specific whole blood gene panels reliably distinguished CD and UC and determined the activity of disease, with high sensitivity and specificity in our cohorts of patients. This simple serological test has the potential to become a biomarker to determine the activity of disease. (Inflamm Bowel Dis 2015;21:1132–1140) Key Words: inflammatory bowel disease, biomarker, gene expression
C
rohn’s disease (CD) and ulcerative colitis (UC) are chronic relapsing inflammatory bowel diseases (IBD) of which the etiologies are not fully understood. Genetic and environmental factors are likely central to both diseases. The clinical course of IBD is characterized by a succession of relapses and remissions. Traditionally, the diagnosis of IBD has been based on radiolog-
Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Web site (www.ibdjournal.org). Received for publication October 22, 2014; Accepted December 22, 2014. From the *Division of Gastroenterology and Hepatology, Department of Medicine, Brigham and Women’s Hospital, Boston, Massachusetts; †Division of Digestive Diseases, David Geffen School of Medicine, UCLA, Los Angeles, California; ‡ Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts; § Sentinel Center, SBC, Shanghai, China; kGoldenHealthDx, Ontario, Canada; and ¶ GeneNews, Toronto, Canada. Supported by a grant from the Broad Medical Research Program to R. Burakoff. The authors have no conflicts of interest to disclose. R. Burakoff and V. Pabby contributed equally to this study. Reprints: Robert Burakoff, MD, MPH, Brigham and Women’s Hospital, 45 Francis Street, Thorn 1428, Boston, MA 02115 (e-mail: [email protected]). Copyright © 2015 Crohn’s & Colitis Foundation of America, Inc. DOI 10.1097/MIB.0000000000000340 Published online 2 April 2015.
1132
| www.ibdjournal.org
2015
ical, endoscopic, and histological examination. After diagnosis, monitoring disease activity and therapeutic response has been ascertained by radiological techniques and colonoscopy. However, endoscopy is invasive, costly, associated with significant risk, and is unable to evaluate the entire gastrointestinal tract. Various serological biomarkers are available including erythrocyte sedimentation rate, C-reactive protein (CRP), p-antineutrophil cytoplasmic antibody (p-ANCA), antisaccharomyces cerevisiae antibody (ASCA), and Escherichia coli outer membrane porin (OmpC).1 The existing serological markers tend to have low sensitivity and specificity because autoimmune diseases, infectious processes, and inflammation outside the gastrointestinal tract may result in elevation of these markers. In contrast, fecal markers, such as calprotectin (FC) and fecal lactoferrin, tend to be more specific for intestinal inflammation. However, increased levels of FC are found in IBD, colonic cancer, in those receiving medications such as nonsteroidal anti-inflammatory drugs, and other conditions.2 Additionally, FC has not been shown to be able to differentiate between CD and UC,3 correlates with colonic disease better than ileal disease, and does not delineate the extent of colonic involvement. Gene expression profiling is being used increasingly to examine the differential expression of genes between disease Inflamm Bowel Dis Volume 21, Number 5, May 2015
states and normal, which can potentially be used in IBD to determine the activity of disease and therapeutic response. Previous techniques included obtaining messenger RNA (mRNA) from patient tissue samples that were hybridized to a gene chip that contained thousands of probes corresponding to individual genes. However, a gene expression analysis from peripheral whole blood is clinically advantageous over biopsy of tissue, as it is minimally invasive and can be performed at any time. More recent advances in this field are the development of techniques to evaluate mRNA from whole blood.4,5 Notably, mRNA gene expression methodology using whole blood has been validated and can be used to stratify an individual into high and low risk for developing colorectal cancer.6 This test can help predict a patient’s current risk of having colorectal cancer.7 We previously published that gene expression profiles from whole blood can differentiate patients with active and inactive CD.8 We also published that gene expression profiles can differentiate between CD, UC, and noninflammatory diarrheal disorders.9 In this study, a supervised learning method (logistic regression) was used and specific panels of 6 probe sets, which achieved discrimination, were identified. Identifying specific genes that are expressed and increased during IBD flares may help stratify disease activity and may be important in the management of these patients in a more personalized manner. Biomarkers that are able to predict levels of inflammation in a precise and reproducible manner are essential in patients with IBD given the poor predictability of the currently available serological biomarkers and clinical disease indices. Using our previous methodology, the aim of this study was to identify panels of genes to determine activity of disease in CD and UC.
MATERIALS AND METHODS Patient Selection The exposure of interest was different disease activity in IBD (mild and moderate-to-severe for CD and UC), and the outcome of interest was a common gene array expression pattern. Patients were recruited from a tertiary referral center, the Center for Crohn’s and Colitis at Brigham and Women’s Hospital (BWH), Boston, MA. Permission to conduct the study was obtained from the Institutional Review Board at BWH (Protocol Number: 2012P001913). For ethical reasons, subjects did not have a colonoscopy solely for the purpose of the study. Subjects were included in the study if they had a colonoscopy to confirm the diagnosis of CD or UC, determine disease activity, or for dysplasia screening. Informed written consent was obtained from potential participants. Once recruited, patients were interviewed and their charts were reviewed to determine baseline demographics and disease characteristics including age, gender, ethnicity, smoking status, duration of IBD, disease location, history of extraintestinal manifestations, current IBD medications, family history of IBD, and history of previous gastrointestinal surgery. In addition, based on the patient interview, the clinical activity scores using
2015
validated indices (Harvey–Bradshaw index [HBI] for CD and Mayo score for UC)10,11 were determined. For CD, HBI scores of ,5 were classified as inactive, 5–7 as mild, and
AND
METHODS
FOR
IBD RESEARCH
Blood-based Biomarkers Used to Predict Disease Activity in Crohn’s Disease and Ulcerative Colitis Robert Burakoff, MD, MPH,* Vikas Pabby, MD, MPH,† Louisa Onyewadume, BA,* Robert Odze, MD,‡ Cheryl Adackapara, MD,‡ Wei Wang, PhD,* Sonia Friedman, MD,* Matthew Hamilton, MD,* Joshua Korzenik, MD,* Jonathan Levine, MD,* Frederick Makrauer, MD,* Changming Cheng, PhD,§ Hai Choo Smith, PhD,k Choong-Chin Liew, PhD,§,k and Samuel Chao, MASC¶
Background: Identifying specific genes that are differentially expressed during inflammatory bowel disease flares may help stratify disease activity. The aim of this study was to identify panels of genes to be able to distinguish disease activity in Crohn’s disease (CD) and ulcerative colitis (UC).
Methods: Patients were grouped into categories based on disease and severity determined by histological grading. Whole blood was collected by PAXgene Blood RNA collection tubes, (PreAnalytiX) and gene expression analysis using messenger RNA was conducted. Logistic regression was performed on multiple combinations of common probe sets, and data were evaluated in terms of discrimination by computing the area under the receiving operator characteristic curve (ROC–AUC). Results: Nine inactive CD, 8 mild CD, 10 moderate-to-severe CD, 9 inactive UC, 8 mild UC, 10 moderate-to-severe UC, and 120 controls were hybridized to Affymetrix U133 Plus 2 microarrays. Panels of 6 individual genes discriminated the stages of disease activity: CD with mild severity {ROC–AUC, 0.89 (95% confidence interval [CI], 0.84%–0.95%)}, CD with moderate-to-severe severity (ROC–AUC 0.98 [95% CI, 0.97–1.0]), UC with mild severity (ROC–AUC 0.92 [95% CI, 0.87–0.96]), and UC with moderate-to-severe severity (ROC–AUC 0.99 [95% CI, 0.97–1.0]). Validation by real-time reverse transcription–PCR confirmed the Affymetrix microarray data.
Conclusions: The specific whole blood gene panels reliably distinguished CD and UC and determined the activity of disease, with high sensitivity and specificity in our cohorts of patients. This simple serological test has the potential to become a biomarker to determine the activity of disease. (Inflamm Bowel Dis 2015;21:1132–1140) Key Words: inflammatory bowel disease, biomarker, gene expression
C
rohn’s disease (CD) and ulcerative colitis (UC) are chronic relapsing inflammatory bowel diseases (IBD) of which the etiologies are not fully understood. Genetic and environmental factors are likely central to both diseases. The clinical course of IBD is characterized by a succession of relapses and remissions. Traditionally, the diagnosis of IBD has been based on radiolog-
Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Web site (www.ibdjournal.org). Received for publication October 22, 2014; Accepted December 22, 2014. From the *Division of Gastroenterology and Hepatology, Department of Medicine, Brigham and Women’s Hospital, Boston, Massachusetts; †Division of Digestive Diseases, David Geffen School of Medicine, UCLA, Los Angeles, California; ‡ Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts; § Sentinel Center, SBC, Shanghai, China; kGoldenHealthDx, Ontario, Canada; and ¶ GeneNews, Toronto, Canada. Supported by a grant from the Broad Medical Research Program to R. Burakoff. The authors have no conflicts of interest to disclose. R. Burakoff and V. Pabby contributed equally to this study. Reprints: Robert Burakoff, MD, MPH, Brigham and Women’s Hospital, 45 Francis Street, Thorn 1428, Boston, MA 02115 (e-mail: [email protected]). Copyright © 2015 Crohn’s & Colitis Foundation of America, Inc. DOI 10.1097/MIB.0000000000000340 Published online 2 April 2015.
1132
| www.ibdjournal.org
2015
ical, endoscopic, and histological examination. After diagnosis, monitoring disease activity and therapeutic response has been ascertained by radiological techniques and colonoscopy. However, endoscopy is invasive, costly, associated with significant risk, and is unable to evaluate the entire gastrointestinal tract. Various serological biomarkers are available including erythrocyte sedimentation rate, C-reactive protein (CRP), p-antineutrophil cytoplasmic antibody (p-ANCA), antisaccharomyces cerevisiae antibody (ASCA), and Escherichia coli outer membrane porin (OmpC).1 The existing serological markers tend to have low sensitivity and specificity because autoimmune diseases, infectious processes, and inflammation outside the gastrointestinal tract may result in elevation of these markers. In contrast, fecal markers, such as calprotectin (FC) and fecal lactoferrin, tend to be more specific for intestinal inflammation. However, increased levels of FC are found in IBD, colonic cancer, in those receiving medications such as nonsteroidal anti-inflammatory drugs, and other conditions.2 Additionally, FC has not been shown to be able to differentiate between CD and UC,3 correlates with colonic disease better than ileal disease, and does not delineate the extent of colonic involvement. Gene expression profiling is being used increasingly to examine the differential expression of genes between disease Inflamm Bowel Dis Volume 21, Number 5, May 2015
states and normal, which can potentially be used in IBD to determine the activity of disease and therapeutic response. Previous techniques included obtaining messenger RNA (mRNA) from patient tissue samples that were hybridized to a gene chip that contained thousands of probes corresponding to individual genes. However, a gene expression analysis from peripheral whole blood is clinically advantageous over biopsy of tissue, as it is minimally invasive and can be performed at any time. More recent advances in this field are the development of techniques to evaluate mRNA from whole blood.4,5 Notably, mRNA gene expression methodology using whole blood has been validated and can be used to stratify an individual into high and low risk for developing colorectal cancer.6 This test can help predict a patient’s current risk of having colorectal cancer.7 We previously published that gene expression profiles from whole blood can differentiate patients with active and inactive CD.8 We also published that gene expression profiles can differentiate between CD, UC, and noninflammatory diarrheal disorders.9 In this study, a supervised learning method (logistic regression) was used and specific panels of 6 probe sets, which achieved discrimination, were identified. Identifying specific genes that are expressed and increased during IBD flares may help stratify disease activity and may be important in the management of these patients in a more personalized manner. Biomarkers that are able to predict levels of inflammation in a precise and reproducible manner are essential in patients with IBD given the poor predictability of the currently available serological biomarkers and clinical disease indices. Using our previous methodology, the aim of this study was to identify panels of genes to determine activity of disease in CD and UC.
MATERIALS AND METHODS Patient Selection The exposure of interest was different disease activity in IBD (mild and moderate-to-severe for CD and UC), and the outcome of interest was a common gene array expression pattern. Patients were recruited from a tertiary referral center, the Center for Crohn’s and Colitis at Brigham and Women’s Hospital (BWH), Boston, MA. Permission to conduct the study was obtained from the Institutional Review Board at BWH (Protocol Number: 2012P001913). For ethical reasons, subjects did not have a colonoscopy solely for the purpose of the study. Subjects were included in the study if they had a colonoscopy to confirm the diagnosis of CD or UC, determine disease activity, or for dysplasia screening. Informed written consent was obtained from potential participants. Once recruited, patients were interviewed and their charts were reviewed to determine baseline demographics and disease characteristics including age, gender, ethnicity, smoking status, duration of IBD, disease location, history of extraintestinal manifestations, current IBD medications, family history of IBD, and history of previous gastrointestinal surgery. In addition, based on the patient interview, the clinical activity scores using
2015
validated indices (Harvey–Bradshaw index [HBI] for CD and Mayo score for UC)10,11 were determined. For CD, HBI scores of ,5 were classified as inactive, 5–7 as mild, and
