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Gastroenterology. Author manuscript; available in PMC 2017 April 01. Published in final edited form as: Gastroenterology. 2016 April ; 150(4): 931–943. doi:10.1053/j.gastro.2015.12.036.
Whole-exome Sequencing analyses of Inflammatory Bowel Disease-associated Colorectal Cancers Ana I. Robles1,*, Giovanni Traverso2,3,*, Ming Zhang4,*, Nicholas J. Roberts4,5, Mohammed A. Khan1, Christine Joseph4, Gregory Y. Lauwers6, Florin M. Selaru7, Maria Popoli4, Meredith E. Pittman5, Xiquan Ke7, Ralph H. Hruban5, Stephen J. Meltzer7, Kenneth W. Kinzler4, Bert Vogelstein4,8, Curtis C. Harris1, and Nickolas Papadopoulos4
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1Laboratory
of Human Carcinogenesis, NCI-CCR, National Institutes of Health, Bethesda, MD,
USA 2Division
of Gastroenterology, Massachusetts General Hospital, Harvard Medical School, Boston,
MA, USA 3Department
of Chemical Engineering and Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA
4Ludwig
Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA
5Department
of Pathology, The Sol Goldman Pancreatic Cancer Research Center, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
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Correspondence: Nickolas Papadopoulos, Department of Oncology, 1650 Orleans Street, CRB1 Room 585, Baltimore, MD 21287, USA ([email protected]), Curtis C. Harris, Laboratory of Human Carcinogenesis, 37 Convent Dr, Rm 3068A, MSC 4258, Bethesda, MD 20892, USA ([email protected]); Bert Vogelstein, Department of Oncology, 1650 Orleans Street, Baltimore, MD 21287, USA ([email protected]). *co-first authors Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Author Contributions AIR, analysis and interpretation of data; drafting of the manuscript GT, analysis and interpretation of data; drafting of the manuscript MZ, acquisition of data NJR, method development and data analysis MAK, technical support CJ, acquisition of data GYL, analysis of data, pathologic technical support FMS, analysis and interpretation of data; drafting of the manuscript MP, acquisition and analysis of data MEP, acquisition and analysis of data XK, acquisition of data and technical support SJM, analysis and interpretation of data; drafting of the manuscript KWK, study concept and design; analysis and interpretation of data BV, study concept and design; drafting of the manuscript CCH, study concept and design; acquisition of data; drafting of the manuscript NP, study concept and design; acquisition of data; analysis and interpretation of data; drafting of the manuscript Author names in bold designate shared co-first authorship Disclosure The authors disclose no personal or financial conflicts
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6Department
of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston,
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MA, USA 7Division
of Gastroenterology, Johns Hopkins University School of Medicine, Baltimore, MD, USA
8Howard
Hughes Medical Institute, and the Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA
Abstract BACKGROUND & AIMS—A long duration of inflammatory bowel disease (IBD) increases the risk for colorectal cancer (CRC). Mutation analysis of limited numbers of genes has indicated that colorectal tumors that develop in patients with IBD differ from those of patients without IBD. We performed whole-exome sequencing analyses to characterize the genetic landscape of these tumors.
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METHODS—We collected colorectal tumor and non-neoplastic tissues from 31 patients with IBD and CRC (15 with ulcerative colitis, 14 with Crohn’s disease, and 2 with indeterminate colitis) and performed whole-exome sequencing analyses of the micro-dissected tumor and matched nontumor tissues. We identified somatic alterations by comparing matched specimens. The prevalence of mutations in sporadic colorectal tumors was obtained from previously published exomesequencing studies.
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RESULTS—Two specimens had somatic mutations in the DNA-proofreading or mismatch repair genes POLE, MLH1, and MSH6 and the tumor cells had a hypermutable phenotype. The remaining tumors had, on average, 71 alterations per sample. TP53 was the most commonly mutated gene, with and incidence prevalence similar to that of sporadic colorectal tumors (63% of cases). However, tumors from the patients with IBD had a different mutation spectrum. APC and KRAS were mutated at significantly lower rates in tumors from patients with IBD than in sporadic colorectal tumors (13% and 20% of cases, respectively). Several genes were mutated more frequently or uniquely in tumors from patients with IBD, including SOX9 and EP300 (which encode proteins in the WNT pathway), NRG1 (which encodes an ERBB ligand), and IL16 (which encodes a cytokine). Our study also revealed recurrent mutations in components of the Rho and Rac GTPase network, indicating a role for non-canonical WNT signaling in development of colorectal tumors in patients with IBD. CONCLUSIONS—Colorectal tumors that develop in patients with IBD have distinct genetic features from sporadic colorectal tumors. These findings could be used to develop disease-specific markers for diagnosis and treatment of patients with IBD and CRC.
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Keywords exome; sequencing; ulcerative colitis; Crohn’s disease
INTRODUCTION Inflammatory bowel disease (IBD), comprising ulcerative colitis (UC) and Crohn’s disease (CD), is associated with an increased risk of developing colorectal cancer (CRC)1–4. Current guidelines for the surveillance of patients with IBD include colonoscopy at frequent
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intervals starting at a maximum of 8 years from the onset of IBD symptoms5, 6. Patients with pancolitis or left-sided colitis are recommended to undergo surveillance colonoscopy every 1–2 years after initial colonoscopy5, 6. Although recent retrospective data show that frequent surveillance reduces CRC incidence and mortality rates in IBD patients7, this represents an intensive, invasive and expensive approach. The burden of intense surveillance could potentially be reduced if a patient’s specific risk for developing CRC could be determined more precisely. No molecular markers are routinely used to stratify IBD patients into groups at low or high risk of developing colonic dysplasia or CRC6. DNA aneuploidy may precede or appear synchronously with dysplasia in both UC8 and CD9 patients.
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The molecular genetics revolution in cancer has led to the identification of the genetic landscapes of numerous tumor types. These findings in turn have enabled the development of diagnostic and prognostic methods10–13 and ushered the era of targeted therapies14. Studies focused on selected regions of the genome suggest that the genetic landscapes of colorectal tumors in patients with and without IBD have both similar and distinct features. For example, IBD-associated colorectal tumors have lower prevalence of APC15–17 and KRAS18–21 mutations than sporadic tumors. Despite this, nuclear beta-catenin accumulation, a hallmark of canonical WNT signaling, is frequently found in colorectal tumors from patients with IBD22, 23. A similar paucity of APC mutations was reported in an animal model of IBD-associated colon cancer24. Several studies suggest that TP53 mutation is a late event in sporadic colorectal tumors, but an early event in IBD-associated tumors25–30. The rate and timing of microsatellite instability (MSI) are similar in IBDassociated and sporadic CRC31–34, as is the prevalence of MLH1 hypermethylation and silencing in MSI-high (MSI-H) neoplasms33, 35–37. However, profiles of coding region microsatellite mutations differ significantly between MSI-H IBD-associated and sporadic CRC34. Similarly, genome-wide mRNA and microRNA expression profiles differ substantially in IBD-associated and sporadic CRC38–45. Moreover, the broad patterns of gene hypermethylation have been shown to be similar46–51 or different37, 52–54 between IBD-associated and sporadic colorectal tumors. These molecular pathologic differences and similarities imply that pathways underlying malignant progression in the two conditions share certain commonalities, but are also largely unique.
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The development of CRC in IBD patients is driven by chronic inflammation, which generates reactive oxygen and nitrogen species. These reactive oxygen and nitrogen species may in turn be the key mutagenic process that underlies the unique molecular features of IBD-associated colorectal tumors55. The presence of specific TP53 mutations in inflamed, but not uninflamed, noncancerous colonic tissue from UC patients30 is one example supporting the promutagenic role of inflammation in IBD. A comprehensive characterization of the mutational landscape of IBD-associated colorectal tumors could provide clues to the etiology of CRC in the context of chronic inflammation, as well as more precisely identify molecular pathways and biomarkers of diagnostic and therapeutic relevance. Herein, we described the results of whole-exome DNA sequencing performed in tumor and paired nontumor colonic tissues from a series of well-characterized IBD patients.
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MATERIALS AND METHODS Whole-Exome Capture and Sequencing and Copy Number Analysis
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DNA samples were purified from archived formalin-fixed paraffin-embedded (FFPE) blocks. Regions of these tumors that were enriched for neoplastic cells (>50%) were microdissected, avoiding foci of inflammation. Library construction was performed with a protocol developed specifically for DNA from archival samples that might contain damaged nucleotides56. The resulting libraries were compatible for sequencing on Illumina instrumentation. Exonic regions were captured in solution using the Agilent SureSelect v.5 kit according to the manufacturer’s instructions (Agilent, Santa Clara, CA) and modifications as published elsewhere57. Next-generation sequencing and bioinformatic analyses were performed at the Goldman Sequencing Center at Johns Hopkins or at Personal Genome Diagnostics (PGDx, Baltimore, MD). Known polymorphisms recorded in dbSNP were removed from the analysis. Potential somatic mutations were filtered and visually inspected as described previously58. Selected somatic mutations were validated using SafeSeqS, a digital method developed for precisely quantifying mutations59. Copy number alterations were identified by comparing normalized average per-base coverage for a particular gene in a tumor sample to the normalized average per-base coverage in the matched normal sample. Mutation prevalence in sporadic CRC was obtained from exomesequencing reported by the Cancer Genome Atlas Research Network60. See the Supplementary Materials and Methods for detailed description of tissue specimens and sequencing, immunohistochemical staining, analysis of mutation patterns and pathway analysis.
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RESULTS Landscape of somatic mutations in IBD-Associated Colon Cancers To generate a census of the genetic alterations that characterize IBD-associated colorectal tumors, we performed whole-exome sequencing on 32 formalin-fixed paraffin-embedded (FFPE) specimens from 31 patients with IBD. These included two patients with indeterminate colitis, 14 CD and 15 UC patients (Supplementary Table 1). An average of 11.6 Gb were sequenced per sample. The average depth of quality coverage of the targeted region was 62-fold (range, 29 to 100-fold), with 76% of targeted bases yielding at least 10 distinct reads (range, 52% – 90%) in the tumors; and 64-fold (range, 17- to 123fold) with at least 78% of targeted bases yielding at least 10 distinct reads in the matched non-tumor samples (Supplementary Table 2).
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Somatic alterations were identified by comparing variants in paired tumor and non-tumor tissues. The number of somatic mutations showed great variability among tumors, with a range of 16 to 1,118 mutations per sample. There were two outlier samples, IBD128 and IBD003, harboring 1,118 and 916 somatic alterations each, with mutation rates of 22.20 and 18.19 mutations/Mb, respectively (Supplementary Figure 1). IBD128, from a CD patient, contained a missense as well as a splice site mutation of the POLE proofreading DNA polymerase and a splice site mutation of POLD1. IBD003, from a UC patient, contained
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single base pair deletions resulting in frameshift mutations of the DNA mismatch repair pathway genes MLH1 and MSH6. The mutation spectrum of these two hypermutated samples was dominated by small deletions, which constituted over 30% of the somatic sequence alterations (Figure 1A; Supplementary Table 3). This profile is consistent with that observed in hypermutated and Microsatellite Instability-High (MSI-H) colorectal60 and gastric61, 62 tumors. After exclusion of these two outliers, the median mutation rate for the remaining colorectal tumors was 1.33 mutations/Mb (range, 0.32 to 5.10 mutations/Mb), with an average of 71 somatic mutations per sample. These mutation rates are similar to those observed in non-hypermutated and Microsatellite-Stable (MSS) samples from other cancers of the GI tract58, 60–64, including those that have inflammatory etiologies, as well as sporadic tumors not readily associated with inflammation (Figure 1B; Supplementary Table 4). The pattern of base changes in non-hypermutated tumors showed a preponderance of C to T transitions (62%), a majority of which (48% of all mutations) occurred at 5′-CpG-3′ sites, a pattern commonly found in other cancers65 and linked to spontaneous deamination of methylated cytosine66 (Figure 2A; Supplementary Figure 2). An excess of A to C transversions at AA dinucleotides, particularly in a context of AAG trinucleotides, was also noted. This kind of enrichment has been described in esophageal67, 68 and gastric62 adenocarcinomas but has not been observed in sporadic CRC (Supplementary Figure 3). There were no apparent differences in the spectrum of substitutions between tumors arising in UC and CD patients (Figures 2B and 2C, respectively) with only a slightly elevated median number of mutations in tumors from UC patients vs. CD patients (77 vs. 53, respectively, excluding the two hypermutated samples). Recurrently mutated genes in IBD-Associated Colon Cancers
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Overall, we identified 4,158 somatic sequence alterations in 3,254 genes, comprising 3,297 nucleotide substitutions, of which 2,997 were missense mutations (Supplementary Table 5). When hypermutated tumors were excluded, we identified 2,124 somatic mutations, 1,953 substitutions and 171 indels in 1,806 genes. Among these, 28 genes showed mutation rates > 10 mutations/Mb and were recurrently mutated in three or more non-hypermutated samples (Table 1). One of the goals of this study was to determine the genomic landscape of colorectal tumors associated with IBD and compare it to that of sporadic CRC, the first tumor type in which genomic landscapes were defined63, 69. To this end, we used the sporadic colorectal tumor landscape recently reported by the Cancer Genome Atlas Research Network as a fiducial60. Although there were similarities between the most commonly mutated genes in both IBD-associated and sporadic CRC, there were also some striking differences (Figure 3; Table 2). APC, the most commonly mutated gene in sporadic colorectal tumors60, was mutated in only four IBD-CRC tumors (13%), a prevalence much lower than expected (Benjamini-Hochberg adjusted Fisher’s exact test P = .0017, Supplementary Table 6). This finding is in agreement with prior single-gene based sequencing efforts15–17. Three of the four IBD-CRC tumors with APC mutation were from CD patients, while the fourth was from a patient with indeterminate colitis. Mutations in KRAS were also less prevalent in IBD-CRC compared to sporadic CRC, again concordant with previous single-gene studies18–21, 60 (adjusted P =.019). No NRAS mutations were found in IBD-CRC tumors, although this gene is often altered in sporadic CRC60. Instead, several genes were identified that were more frequently or uniquely mutated in IBD-CRC,
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including IL16, RADIL, RRBP1, SRRM4, DOCK3, TRRAP, CACNA1D, NRG1, EP300, and RIMS2 (Figure 3; Supplementary Table 6).
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We identified four tumors with IL16 mutations. Interleukin-16 is a chemoattractant cytokine that can stimulate the secretion of tumor-associated inflammatory cytokines by monocytes70, thus supporting a sustained inflammatory response. NRG1, encoding a ligand for the ERBB3 and ERBB4 tyrosine kinase receptors71 was also recurrently mutated, as were genes coding for RAC1 guanine nucleotide exchange factors (GEFs), including two members of the dedicator of cytokinesis (Dock) family, DOCK2 and DOCK3, as well as PREX2. Recent exome sequencing studies have revealed recurrent DOCK2 mutations in esophageal67, 72 and colon73 cancers, and recurrent PREX2 mutations in melanoma74. We also found novel somatic mutations in RADIL, which, together with its paralog RASIP1, controls Rho signaling associated with cell spreading75. Additionally, we observed recurrent mutations in EP300 and TRRAP, which function in chromatin remodeling through histone acetylation and which facilitate p53-mediated transcription76. TP53 was the most commonly mutated gene (19/30 cases, 63%), with mutation prevalence similar to that in sporadic CRC (Table 2; Figure 3). Other genes similarly mutated in IBD-CRC and sporadic CRC included SMAD2/4, PIK3CA, SOX9, FBXW7, and TCF7L2 (Table 2). All three PIK3CA mutations occurred at hotspots previously associated with CRC and other cancers. Likely oncogenic driver gene mutations in BRAF, CTNNB1, and CREBBP were found in one IBD-CRC case each (CHASM scores < 0.2).
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The mutation spectrum of TP53 is of particular interest in light of its prevalence and documented association with environmental exposures77. As expected, the majority of TP53 mutations (14/19) observed in IBD-CRC tumors were missense (Supplementary Table 7), and located predominantly in the DNA binding domain of the protein (aa.100–300, Figure 4). However, there were some notable differences in the spectrum of TP53 single substitutions in IBD-associated neoplasia and sporadic CRC. Specifically, no mutations were observed at the hotspot R273, and only one mutation was found at each of the hotspots R248, G245 and R175, in IBD-associated colorectal tumors. Instead, three of the 19 tumors harbored the less common R282W mutation, and two tumors each had mutations in R158, H179, or R342, which are rarely found in sporadic CRC (Supplementary Table 7). TP53 alterations were found in eight UC- and nine CD-associated tumors. In IBD-associated colorectal tumors, the predominant substitution was a C:G>T:A transition at CpG dinucleotides (n=10, 52.6%). This type of substitution in TP53 has previously been positively correlated with expression of the enzyme inducible NO synthase (iNOS)78 in colonic tumors, where it has been hypothesized to be readout of inflammation-associated DNA damage. Indeed, colonic iNOS expression is known to be increased in IBD79, 80.
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Copy Number Alterations in IBD-Associated CRCs We identified 15 regions with at least five-fold amplification many of which were recurrent or had clinically-relevant genes within them (Supplementary Table 8). These included 8q24.21, containing the MYC oncogene, and 17q12, involving ERBB2, as previously reported in sporadic CRC60, 81. CDX2, a homeobox transcription factor involved in intestinal development82, was the most commonly amplified gene. Recurrently amplified
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regions containing IRS2, involved in activation of the PI3K pathway and recently identified as a candidate driver of CRC83, or containing Suppressor of Cytokine Signaling 1 (SOCS1), were also identified. The only recurrently deleted region spanned the HLA-DQB1 locus in 6p21, a region previously associated with UC risk84. Interestingly, this alteration was found in 6 patients with UC, 1 patient with CD and 1 patient with indeterminate colitis. Pathways Altered in IBD-CRC The set of mutated genes was enriched for gene ontologies associated with cell communication, cell-cell signaling, and cell adhesion (Supplementary Table 9). These findings are consistent with dysregulated cytokines and inflammatory mediators playing a role in IBD-associated CRC.
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We grouped mutated genes into pathways and functions to better understand the overall consequences of mutations in IBD-CRC and how the pathways and functions altered in IBD-CRC compare to those altered in sporadic CRC. We found recurrent alterations in the p53, RTK, RAS, PI3K, WNT, and TGF-ß pathways, as has been previously described for sporadic CRC60. In addition to these established CRC-associated pathways, genes involved in the Rho GTPase network, cell communication/adhesion, cytokine signaling, and chromatin remodeling were recurrently altered in IBD-CRC.
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WNT signaling pathway alterations involving inactivation of APC or activation of CTNNB1 were found in only 17% (5/30) non-hypermutated IBD-CRC tumors, however, recurrent mutations were found on EP300 and SOX9, also thought to be involved in WNT signaling. Several other genes in this pathway, including FZD8, AXIN1, TCF7L2, NKD2, MAP3K7, NLK, ARID1A, and CREBBP harbored private mutations (Supplementary Figure 4). Even considering these, only 53% of the IBD-CRCs had mutations in the WNT signaling pathway, a much lower proportion than has been reported in sporadic CRC60. To directly evaluate the status of WNT signaling in IBD-CRC, we performed beta-catenin immunohistochemical staining in 17 tumors with available material (Supplementary Figure 5; Supplementary Table 10). Not surprisingly, two tumors with APC inactivating and one with CTNNB1 activating mutations showed nuclear accumulation of beta-catenin. TCF7L2 and SOX9 are also considered part of the WNT canonical pathway and two tumors with inactivating mutations in these genes also showed beta-catenin nuclear accumulation. In addition, two tumors without obvious mutations in genes that are part of the canonical WNT pathway showed beta-catenin nuclear staining, although we can’t exclude large deletions of APC that would be missed by exome sequencing. Thus, despite the lower rate of inactivating APC or activating CTNNB1 mutations, the WNT pathway is still dysregulated in IBD-CRC through activating mutations in other components of the pathway. However, the rate of 41% nuclear accumulation of beta-catenin is still much lower than that in sporadic CRCs. The overall rate of mutation in TGF-ß signaling pathway was similar to that of sporadic CRC60. We found SMAD2/4 mutations in 7 tumors (23%), more commonly than in a previous single gene study85. Infrequent mutations of other genes in this pathway, including solitary mutations of TGFBR2, ACVR1, ACVR2B, and SMAD3, were identified in IBD-CRCs (Supplementary Figure 4). Recurrent alterations in genes involved in receptor-mediated signaling pathways were identified in 60% of IBD-CRCs (Supplementary Figure 4). These
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included mutually exclusive amplification of IRS2 and ERBB2, and mutually exclusive mutations of PIK3CA and KRAS, as well as NRG1 mutations.
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Four recurrently mutated genes, DOCK2, DOCK3, PREX2, and RADIL, belonging to the Rho and Rac GTPase network, were mutated in 30% of IBD-CRCs (9/30). Perusing the list of genes with at least one non-silent somatic mutation, we found mutations in several other activating RhoGEFs, along with inactivating RhoGTPase-activating proteins (RhoGAPs)86. Overall, the Rho and Rac pathway was altered in 50% of cases (Supplementary Figure 4). Moreover, a striking 50% of cases (15/30) presented mutations in chromatin remodeling genes, including recurrent mutations in TRRAP and EP300, and low-frequency mutations in CREBBP, ARID1A, and others (Supplementary Figure 4). Dysregulation of cytokine signaling is characteristic of IBD87. We found recurrent mutually exclusive mutations in IL-16 and amplification of SOCS1 in 7 (24%) of cases (Supplementary Figure 4). Genetic alterations functionally associated with cell-cell communication or adhesion were found in 60% of IBD-CRCs, owing to recurrent alterations in CSMD2/3, DOCK2/3, CTNND2, DSCAML1, PTPRF and RADIL (Supplementary Figure 4). Comparison of genes and pathways altered in UC and CD-associated CRC
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Although both UC and CD patients are at elevated risk of developing CRC compared to the general population, the incidence is higher in patients with extensive colitis88. This may be related to more sustained and pervasive inflammation of the colonic mucosa in UC patients. We looked at the overall involvement of each major pathway identified per tumor and compared how these pathways were differentially altered in UC vs. CD (Figure 5). We found that the Rho and Rac pathway was affected in 10 CRCs from confirmed UC patients but in only 3 CRCs from confirmed CD patients (Fisher’s exact test P = .025). With the caveat of our relatively small sample size, it appears that the Rho and Rac pathway may be preferentially activated in UC patients.
DISCUSSION
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IBD patients experience a higher incidence of CRC than the general population. Studies involving limited numbers of genes have suggested that chronic inflammation-driven CRC possesses unique genetic features. Herein, we have presented a comprehensive characterization of the genomic landscape of IBD-CRC and compared it to that of sporadic colorectal tumors. We identified a number of similarities between IBD-associated and sporadic colorectal tumors, indicating that the development of these tumors involves, for the most part, the same cellular pathways. However, there are striking differences. Mutations in key genes involved in the development and progression of sporadic CRC are uncommonly altered in IBD-CRC, suggesting an underlying difference in mechanism or etiology between IBD-associated and sporadic colorectal tumors. Almost all sporadic colorectal tumors have altered WNT pathway genes, most commonly APC. By contrast, we found a lower rate of APC inactivation, but a higher relative number of inactivating mutations in the SOX9 transcription factor, an intestinal stem cell marker89 that antagonizes WNT/beta-catenin signaling90. It has been previously suggested that WNT signaling can be epigenetically activated in IBD-CRC51, thus bypassing the need for APC Gastroenterology. Author manuscript; available in PMC 2017 April 01.
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mutations. Moreover, the WNT pathway could be activated by inflammation in the absence of APC or CTNNB1 mutations, possibly through NF-kB and STAT355. These non-genetic pathways need to be formally explored experimentally. Additionally, we discovered recurrent mutations in components of the Rho and Rac GTPase network and cytokine signaling that support a role for non-canonical WNT signaling in IBD-associated neoplasia. Guanine nucleotide exchange factors (GEFs) promote activation of the Rho GTPase Rac1, which in the colon mediates the production of reactive oxygen species and activation of NFκB, leading to stimulation of WNT signaling and expansion of the intestinal stem cell pool91. Interestingly, abrogation of Rac1 activity pharmacologically92 or genetically91 in animal models prevents the development of colon cancer, suggesting a potential therapeutic benefit from targeting the Rho and Rac pathway in IBD.
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Similar to APC, the prevalence of KRAS mutations in IBD-CRCs was much lower than in sporadic colorectal tumors, while this was not true for SMAD4 and PIK3CA. Approximately 60% of IBD-CRCs had mutations in TP53, a rate equivalent to that of sporadic colorectal tumors. However, hotspot mutations were different between the IBDCRCs and sporadic colorectal tumors. Prior work has suggested that TP53 mutations are acquired early in the IBD-CRC tumorigenic process whereas in sporadic CRCs they have been observed late in the transition from adenoma to carcinoma93, 94. We observed a TP53 mutation rate matching that of sporadic CRCs potentially arguing against this prior observation. However, our study did not include precancerous lesions; therefore, our data is inconclusive regarding the timing of mutations in IBD-CRC. Future studies that evaluate a larger number IBD-CRCs at various stages of development will be required to help delineate this process better.
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A number of mutations and copy number alterations in targetable oncogenic driver genes with well-established ties to sporadic CRC were also found in IBD-CRC. These included hotspot driver mutations in PIK3CA and BRAF, and amplification of IRS2 and ERBB2.
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The presence of recurrent mutations in interleukin-16 (IL16) is particularly interesting, as this cytokine is known to aggravate immune-mediated and autoimmune inflammatory conditions, including IBD95. Serum levels of IL-16 are higher in CRC patients and IL16 gene polymorphisms are associated with susceptibility to CRC96. Expression of IL-16 is upregulated in the colonic mucosae of IBD patients97, 98, and its neutralization reduces injury and inflammation in an animal model of colitis97. Although predictions regarding the functional effect of substitutions are limited by the lack of reliable 3-D models and computational methods to assess most of the uncommon mutations, the available predictions would suggest that mutations in IL-16 could have damaging consequences (Supplementary Table 5). Further studies are required to experimentally determine their specific functional effect. Complementary to our study, sequencing approaches that comprehensively determine mutations in introns, and long noncoding RNA’s, and molecular studies that evaluate epigenetic silencing of tumor suppressors/activation of oncogenes will be an important next step, which should provide further insight into the molecular changes that promote colorectal tumorigenesis in patients with IBD.
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Our finding of a pattern of base changes at AA dinucleotides resembling that found in esophageal and gastric cancers, both of which are tied to chronic inflammation, provides clues to a putative mutagenic role of inflammation that also merits further scrutiny. In summary, this in-depth analysis of colorectal tumors associated with IBD demonstrates a unique molecular profile and provides clues to the etiology of CRC in IBD patients. Our study also sets the stage for improved early detection of colorectal tumors in IBD patients based on their unique genetic composition, as well as for the tailoring of therapies in this patient population.
Supplementary Material Refer to Web version on PubMed Central for supplementary material.
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Acknowledgments We thank Ms. Laura Nakatsuka and Mr Barrett Goodspeed from the MGH Pathology Department for their assistance in specimen identification and procurement. Grant Support This work was supported by the Intramural Research Program of the National Cancer Institute, NIH, Virginia and D.K. Ludwig Fund for Cancer Research, The Sol Goldman Sequencing Facility at Johns Hopkins, and NIH Grants CA43460 (NP), CA057345 (NP), CA152753 (NP), T32-DK007191-38-S1 (GT), CA62924 (RHH), CA133012 (SJM), and CA190040 (SJM & FMS).
Abbreviations Author Manuscript
CRC
colorectal cancer
FFPE
formalin-fixed paraffin-embedded
GEF
Guanine nucleotide exchange factor
GWAS
genome-wide association studies
MSI
microsatellite instability
MSS
Microsatellite-Stable
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Figure 1.
A. Number of genetic alterations (mutations, small insertions/deletions, focal amplifications) detected through sequencing and copy number analyses in each of 32 IBD-associated tumors. Samples are organized by descending total number of alterations and type of IBD. B. Median number of somatic mutations per tumor in non-hypermutated IBD-Colorectal (this study, red frame) and in other tumors of the GI tract. Horizontal bars indicate the 25 and 75% quartiles. MSI, microsatellite instability; hypermut, hypermutable phenotype; EAC, esophageal adenocarcinoma; MSS, microsatellite stable; ESCC, esophageal squamous cell
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carcinoma. Published data on which this figure is based are provided in Supplementary Table 4.
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Author Manuscript Author Manuscript Author Manuscript Figure 2.
Author Manuscript
A. Mutation sequence context of non-hypermutated IBD-associated tumors. Base substitutions were collated into categories representing the 6 possible base changes (represented by colors in the upper right) and further subdivided into the 16 possible combinations that take into account the identity of nucleotides flanking the mutated base, or “trinucleotide context” (for more detail see Supplementary Figure 3). The fractional breakdown of mutation counts is shown in the pie chart on the upper left. B. Mutation sequence context of non-hypermutated tumors from UC cases. C. Mutation sequence context of non-hypermutated tumors from CD cases.
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Figure 3.
Genes recurrently mutated in IBD-associated CRC and their prevalence in sporadic CRC (TCGA). Each column denotes an individual tumor and each row represents a gene. Depicted are genes with mutation rates > 10 mutations/Mb, colored by the type of coding mutation, and marked by * if present in COSMIC. Right, percent of mutated cases in this study (black bars) and in sporadic CRC from TCGA (gray bars). Genes marked by * show mutation prevalence different from that of CRC (two-tailed Fisher’s exact test with
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Benjamini-Hochberg correction for multiple testing, P < .05). Bottom, recurrently-amplified genes and private hotspot mutations.
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Figure 4.
TP53 mutation spectrum in IBD-associated CRC. A. Mutation signature of 19 IBDassociated tumors with TP53 mutations compared to that of all somatic TP53 mutations in human sporadic CRC obtained from the IARC TP53 database (http://p53.iarc.fr/). B. Distribution of TP53 single nucleotide substitutions in IBD-associated CRC. C. Distribution of TP53 single nucleotide substitutions in sporadic CRC obtained from the IARC TP53 database. D. Distribution of TP53 single nucleotide substitutions in sporadic CRC identified
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by exome sequencing in the TCGA study, obtained from cBioPortal (http:// www.cbioportal.org/).
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Author Manuscript Author Manuscript Figure 5.
Author Manuscript
Signaling pathways affected in CD and UC. Genes recurrently mutated or amplified were assigned to manually curated pathways based on KEGG, Gene Ontology, Pathway Commons, and Ingenuity Pathway Analysis (IPA Ingenuity Systems, http:// www.ingenuity.com). Pathways for discussion were selected based on prior association with CRC or the presence of multiple recurrently mutated genes in the pathway.
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Author Manuscript
Author Manuscript 3 3 4 3 4 4 3 3 3 3 3 4 3 5 3 3 3 3 5 5 3 3 4 3
SMAD2
SOX9
NRG1
TPO
IL16
RIMS2
PIK3CA
RADIL
CTNND2
RRBP1
ZCCHC6
DOCK3
PREX2
APC
DOCK2
PTPRF
DSCAML1
CACNA1D
CSMD3
TRRAP
EP300
MTOR
CSMD2
UBR5
4
SMAD4 4
6
SRRM4
19
KRAS
No. of mutations
TP53
Gene symbol
Gastroenterology. Author manuscript; available in PMC 2017 April 01. 3
4
3
3
4
5
3
3
3
3
4
3
4
3
3
3
3
3
4
4
3
4
3
3
3
4
6
19
No. of mutated samples
10%
13%
10%
10%
13%
17%
10%
10%
10%
10%
13%
10%
13%
10%
10%
10%
10%
10%
13%
13%
10%
13%
10%
10%
10%
13%
20%
63%
Percent of cases altered
8400
10896
7650
7245
11580
11124
6546
6342
5724
5493
8532
4821
6093
4488
4233
3678
3228
3207
4236
3999
2802
2142
1530
1404
1836
1659
570
1182
Gene size (bp)
12
12
13
14
14
15
15
16
17
18
20
21
22
22
24
27
31
31
31
33
36
62
65
71
73
80
351
536
Mut / Mb
Yes
–
Yes
Yes
Yes
Yes
Yes
–
Yes
Yes
Yes
Yes
Yes
–
–
–
–
Yes
Yes
–
Yes
–
–
Yes
–
Yes
Yes
Yes
Mutation in Biologically Relevant Gene
Author Manuscript
Recurrently mutated genes in 30 non-hypermutated IBD-associated tumors
–
–
Yes
Yes
–
–
–
–
–
–
Yes
–
–
–
–
–
–
Yes
–
–
–
–
–
Yes
–
Yes
Yes
Yes
Mutation in Clinically Relevant Gene
Yes
–
–
Yes
Yes
–
Yes
–
–
–
Yes
–
–
–
–
–
–
Yes
–
–
–
Yes
–
–
–
Yes
Yes
Yes
Mutation in COSMIC Cancer Gene Census
Author Manuscript
Table 1 Robles et al. Page 24
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Table 2
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Recurrently mutated genes in non-hypermutated sporadic CRC and their mutation frequency in IBD-CRC
Author Manuscript
Percent of sporadic CRC cases altereda
Percent of IBD-CRC cases altered
P valueb
APC
81%
13%
0.0001
TP53
60%
63%
NS
KRAS
43%
20%
0.016
PIK3CA
18%
10%
NS
FBXW7
11%
7%
NS
SMAD4
10%
13%
NS
NRAS
9%
0%
NS
TCF7L2
9%
7%
NS
FAM123B
7%
0%
NS
SMAD2
6%
10%
NS
CTNNB1
5%
3%
NS
KIAA1804
4%
0%
NS
ACVR1B
4%
0%
NS
GPC6
4%
3%
NS
SOX9
4%
10%
NS
EDNRB
3%
0%
NS
Gene symbol
a
Sporadic CRC mutation profile derived from TCGA.
b
Based on Fisher’s Exact test
NS, not significant
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Gastroenterology. Author manuscript; available in PMC 2017 April 01. Published in final edited form as: Gastroenterology. 2016 April ; 150(4): 931–943. doi:10.1053/j.gastro.2015.12.036.
Whole-exome Sequencing analyses of Inflammatory Bowel Disease-associated Colorectal Cancers Ana I. Robles1,*, Giovanni Traverso2,3,*, Ming Zhang4,*, Nicholas J. Roberts4,5, Mohammed A. Khan1, Christine Joseph4, Gregory Y. Lauwers6, Florin M. Selaru7, Maria Popoli4, Meredith E. Pittman5, Xiquan Ke7, Ralph H. Hruban5, Stephen J. Meltzer7, Kenneth W. Kinzler4, Bert Vogelstein4,8, Curtis C. Harris1, and Nickolas Papadopoulos4
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1Laboratory
of Human Carcinogenesis, NCI-CCR, National Institutes of Health, Bethesda, MD,
USA 2Division
of Gastroenterology, Massachusetts General Hospital, Harvard Medical School, Boston,
MA, USA 3Department
of Chemical Engineering and Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA
4Ludwig
Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA
5Department
of Pathology, The Sol Goldman Pancreatic Cancer Research Center, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
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Correspondence: Nickolas Papadopoulos, Department of Oncology, 1650 Orleans Street, CRB1 Room 585, Baltimore, MD 21287, USA ([email protected]), Curtis C. Harris, Laboratory of Human Carcinogenesis, 37 Convent Dr, Rm 3068A, MSC 4258, Bethesda, MD 20892, USA ([email protected]); Bert Vogelstein, Department of Oncology, 1650 Orleans Street, Baltimore, MD 21287, USA ([email protected]). *co-first authors Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Author Contributions AIR, analysis and interpretation of data; drafting of the manuscript GT, analysis and interpretation of data; drafting of the manuscript MZ, acquisition of data NJR, method development and data analysis MAK, technical support CJ, acquisition of data GYL, analysis of data, pathologic technical support FMS, analysis and interpretation of data; drafting of the manuscript MP, acquisition and analysis of data MEP, acquisition and analysis of data XK, acquisition of data and technical support SJM, analysis and interpretation of data; drafting of the manuscript KWK, study concept and design; analysis and interpretation of data BV, study concept and design; drafting of the manuscript CCH, study concept and design; acquisition of data; drafting of the manuscript NP, study concept and design; acquisition of data; analysis and interpretation of data; drafting of the manuscript Author names in bold designate shared co-first authorship Disclosure The authors disclose no personal or financial conflicts
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6Department
of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston,
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MA, USA 7Division
of Gastroenterology, Johns Hopkins University School of Medicine, Baltimore, MD, USA
8Howard
Hughes Medical Institute, and the Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA
Abstract BACKGROUND & AIMS—A long duration of inflammatory bowel disease (IBD) increases the risk for colorectal cancer (CRC). Mutation analysis of limited numbers of genes has indicated that colorectal tumors that develop in patients with IBD differ from those of patients without IBD. We performed whole-exome sequencing analyses to characterize the genetic landscape of these tumors.
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METHODS—We collected colorectal tumor and non-neoplastic tissues from 31 patients with IBD and CRC (15 with ulcerative colitis, 14 with Crohn’s disease, and 2 with indeterminate colitis) and performed whole-exome sequencing analyses of the micro-dissected tumor and matched nontumor tissues. We identified somatic alterations by comparing matched specimens. The prevalence of mutations in sporadic colorectal tumors was obtained from previously published exomesequencing studies.
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RESULTS—Two specimens had somatic mutations in the DNA-proofreading or mismatch repair genes POLE, MLH1, and MSH6 and the tumor cells had a hypermutable phenotype. The remaining tumors had, on average, 71 alterations per sample. TP53 was the most commonly mutated gene, with and incidence prevalence similar to that of sporadic colorectal tumors (63% of cases). However, tumors from the patients with IBD had a different mutation spectrum. APC and KRAS were mutated at significantly lower rates in tumors from patients with IBD than in sporadic colorectal tumors (13% and 20% of cases, respectively). Several genes were mutated more frequently or uniquely in tumors from patients with IBD, including SOX9 and EP300 (which encode proteins in the WNT pathway), NRG1 (which encodes an ERBB ligand), and IL16 (which encodes a cytokine). Our study also revealed recurrent mutations in components of the Rho and Rac GTPase network, indicating a role for non-canonical WNT signaling in development of colorectal tumors in patients with IBD. CONCLUSIONS—Colorectal tumors that develop in patients with IBD have distinct genetic features from sporadic colorectal tumors. These findings could be used to develop disease-specific markers for diagnosis and treatment of patients with IBD and CRC.
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Keywords exome; sequencing; ulcerative colitis; Crohn’s disease
INTRODUCTION Inflammatory bowel disease (IBD), comprising ulcerative colitis (UC) and Crohn’s disease (CD), is associated with an increased risk of developing colorectal cancer (CRC)1–4. Current guidelines for the surveillance of patients with IBD include colonoscopy at frequent
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intervals starting at a maximum of 8 years from the onset of IBD symptoms5, 6. Patients with pancolitis or left-sided colitis are recommended to undergo surveillance colonoscopy every 1–2 years after initial colonoscopy5, 6. Although recent retrospective data show that frequent surveillance reduces CRC incidence and mortality rates in IBD patients7, this represents an intensive, invasive and expensive approach. The burden of intense surveillance could potentially be reduced if a patient’s specific risk for developing CRC could be determined more precisely. No molecular markers are routinely used to stratify IBD patients into groups at low or high risk of developing colonic dysplasia or CRC6. DNA aneuploidy may precede or appear synchronously with dysplasia in both UC8 and CD9 patients.
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The molecular genetics revolution in cancer has led to the identification of the genetic landscapes of numerous tumor types. These findings in turn have enabled the development of diagnostic and prognostic methods10–13 and ushered the era of targeted therapies14. Studies focused on selected regions of the genome suggest that the genetic landscapes of colorectal tumors in patients with and without IBD have both similar and distinct features. For example, IBD-associated colorectal tumors have lower prevalence of APC15–17 and KRAS18–21 mutations than sporadic tumors. Despite this, nuclear beta-catenin accumulation, a hallmark of canonical WNT signaling, is frequently found in colorectal tumors from patients with IBD22, 23. A similar paucity of APC mutations was reported in an animal model of IBD-associated colon cancer24. Several studies suggest that TP53 mutation is a late event in sporadic colorectal tumors, but an early event in IBD-associated tumors25–30. The rate and timing of microsatellite instability (MSI) are similar in IBDassociated and sporadic CRC31–34, as is the prevalence of MLH1 hypermethylation and silencing in MSI-high (MSI-H) neoplasms33, 35–37. However, profiles of coding region microsatellite mutations differ significantly between MSI-H IBD-associated and sporadic CRC34. Similarly, genome-wide mRNA and microRNA expression profiles differ substantially in IBD-associated and sporadic CRC38–45. Moreover, the broad patterns of gene hypermethylation have been shown to be similar46–51 or different37, 52–54 between IBD-associated and sporadic colorectal tumors. These molecular pathologic differences and similarities imply that pathways underlying malignant progression in the two conditions share certain commonalities, but are also largely unique.
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The development of CRC in IBD patients is driven by chronic inflammation, which generates reactive oxygen and nitrogen species. These reactive oxygen and nitrogen species may in turn be the key mutagenic process that underlies the unique molecular features of IBD-associated colorectal tumors55. The presence of specific TP53 mutations in inflamed, but not uninflamed, noncancerous colonic tissue from UC patients30 is one example supporting the promutagenic role of inflammation in IBD. A comprehensive characterization of the mutational landscape of IBD-associated colorectal tumors could provide clues to the etiology of CRC in the context of chronic inflammation, as well as more precisely identify molecular pathways and biomarkers of diagnostic and therapeutic relevance. Herein, we described the results of whole-exome DNA sequencing performed in tumor and paired nontumor colonic tissues from a series of well-characterized IBD patients.
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MATERIALS AND METHODS Whole-Exome Capture and Sequencing and Copy Number Analysis
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DNA samples were purified from archived formalin-fixed paraffin-embedded (FFPE) blocks. Regions of these tumors that were enriched for neoplastic cells (>50%) were microdissected, avoiding foci of inflammation. Library construction was performed with a protocol developed specifically for DNA from archival samples that might contain damaged nucleotides56. The resulting libraries were compatible for sequencing on Illumina instrumentation. Exonic regions were captured in solution using the Agilent SureSelect v.5 kit according to the manufacturer’s instructions (Agilent, Santa Clara, CA) and modifications as published elsewhere57. Next-generation sequencing and bioinformatic analyses were performed at the Goldman Sequencing Center at Johns Hopkins or at Personal Genome Diagnostics (PGDx, Baltimore, MD). Known polymorphisms recorded in dbSNP were removed from the analysis. Potential somatic mutations were filtered and visually inspected as described previously58. Selected somatic mutations were validated using SafeSeqS, a digital method developed for precisely quantifying mutations59. Copy number alterations were identified by comparing normalized average per-base coverage for a particular gene in a tumor sample to the normalized average per-base coverage in the matched normal sample. Mutation prevalence in sporadic CRC was obtained from exomesequencing reported by the Cancer Genome Atlas Research Network60. See the Supplementary Materials and Methods for detailed description of tissue specimens and sequencing, immunohistochemical staining, analysis of mutation patterns and pathway analysis.
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RESULTS Landscape of somatic mutations in IBD-Associated Colon Cancers To generate a census of the genetic alterations that characterize IBD-associated colorectal tumors, we performed whole-exome sequencing on 32 formalin-fixed paraffin-embedded (FFPE) specimens from 31 patients with IBD. These included two patients with indeterminate colitis, 14 CD and 15 UC patients (Supplementary Table 1). An average of 11.6 Gb were sequenced per sample. The average depth of quality coverage of the targeted region was 62-fold (range, 29 to 100-fold), with 76% of targeted bases yielding at least 10 distinct reads (range, 52% – 90%) in the tumors; and 64-fold (range, 17- to 123fold) with at least 78% of targeted bases yielding at least 10 distinct reads in the matched non-tumor samples (Supplementary Table 2).
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Somatic alterations were identified by comparing variants in paired tumor and non-tumor tissues. The number of somatic mutations showed great variability among tumors, with a range of 16 to 1,118 mutations per sample. There were two outlier samples, IBD128 and IBD003, harboring 1,118 and 916 somatic alterations each, with mutation rates of 22.20 and 18.19 mutations/Mb, respectively (Supplementary Figure 1). IBD128, from a CD patient, contained a missense as well as a splice site mutation of the POLE proofreading DNA polymerase and a splice site mutation of POLD1. IBD003, from a UC patient, contained
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single base pair deletions resulting in frameshift mutations of the DNA mismatch repair pathway genes MLH1 and MSH6. The mutation spectrum of these two hypermutated samples was dominated by small deletions, which constituted over 30% of the somatic sequence alterations (Figure 1A; Supplementary Table 3). This profile is consistent with that observed in hypermutated and Microsatellite Instability-High (MSI-H) colorectal60 and gastric61, 62 tumors. After exclusion of these two outliers, the median mutation rate for the remaining colorectal tumors was 1.33 mutations/Mb (range, 0.32 to 5.10 mutations/Mb), with an average of 71 somatic mutations per sample. These mutation rates are similar to those observed in non-hypermutated and Microsatellite-Stable (MSS) samples from other cancers of the GI tract58, 60–64, including those that have inflammatory etiologies, as well as sporadic tumors not readily associated with inflammation (Figure 1B; Supplementary Table 4). The pattern of base changes in non-hypermutated tumors showed a preponderance of C to T transitions (62%), a majority of which (48% of all mutations) occurred at 5′-CpG-3′ sites, a pattern commonly found in other cancers65 and linked to spontaneous deamination of methylated cytosine66 (Figure 2A; Supplementary Figure 2). An excess of A to C transversions at AA dinucleotides, particularly in a context of AAG trinucleotides, was also noted. This kind of enrichment has been described in esophageal67, 68 and gastric62 adenocarcinomas but has not been observed in sporadic CRC (Supplementary Figure 3). There were no apparent differences in the spectrum of substitutions between tumors arising in UC and CD patients (Figures 2B and 2C, respectively) with only a slightly elevated median number of mutations in tumors from UC patients vs. CD patients (77 vs. 53, respectively, excluding the two hypermutated samples). Recurrently mutated genes in IBD-Associated Colon Cancers
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Overall, we identified 4,158 somatic sequence alterations in 3,254 genes, comprising 3,297 nucleotide substitutions, of which 2,997 were missense mutations (Supplementary Table 5). When hypermutated tumors were excluded, we identified 2,124 somatic mutations, 1,953 substitutions and 171 indels in 1,806 genes. Among these, 28 genes showed mutation rates > 10 mutations/Mb and were recurrently mutated in three or more non-hypermutated samples (Table 1). One of the goals of this study was to determine the genomic landscape of colorectal tumors associated with IBD and compare it to that of sporadic CRC, the first tumor type in which genomic landscapes were defined63, 69. To this end, we used the sporadic colorectal tumor landscape recently reported by the Cancer Genome Atlas Research Network as a fiducial60. Although there were similarities between the most commonly mutated genes in both IBD-associated and sporadic CRC, there were also some striking differences (Figure 3; Table 2). APC, the most commonly mutated gene in sporadic colorectal tumors60, was mutated in only four IBD-CRC tumors (13%), a prevalence much lower than expected (Benjamini-Hochberg adjusted Fisher’s exact test P = .0017, Supplementary Table 6). This finding is in agreement with prior single-gene based sequencing efforts15–17. Three of the four IBD-CRC tumors with APC mutation were from CD patients, while the fourth was from a patient with indeterminate colitis. Mutations in KRAS were also less prevalent in IBD-CRC compared to sporadic CRC, again concordant with previous single-gene studies18–21, 60 (adjusted P =.019). No NRAS mutations were found in IBD-CRC tumors, although this gene is often altered in sporadic CRC60. Instead, several genes were identified that were more frequently or uniquely mutated in IBD-CRC,
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including IL16, RADIL, RRBP1, SRRM4, DOCK3, TRRAP, CACNA1D, NRG1, EP300, and RIMS2 (Figure 3; Supplementary Table 6).
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We identified four tumors with IL16 mutations. Interleukin-16 is a chemoattractant cytokine that can stimulate the secretion of tumor-associated inflammatory cytokines by monocytes70, thus supporting a sustained inflammatory response. NRG1, encoding a ligand for the ERBB3 and ERBB4 tyrosine kinase receptors71 was also recurrently mutated, as were genes coding for RAC1 guanine nucleotide exchange factors (GEFs), including two members of the dedicator of cytokinesis (Dock) family, DOCK2 and DOCK3, as well as PREX2. Recent exome sequencing studies have revealed recurrent DOCK2 mutations in esophageal67, 72 and colon73 cancers, and recurrent PREX2 mutations in melanoma74. We also found novel somatic mutations in RADIL, which, together with its paralog RASIP1, controls Rho signaling associated with cell spreading75. Additionally, we observed recurrent mutations in EP300 and TRRAP, which function in chromatin remodeling through histone acetylation and which facilitate p53-mediated transcription76. TP53 was the most commonly mutated gene (19/30 cases, 63%), with mutation prevalence similar to that in sporadic CRC (Table 2; Figure 3). Other genes similarly mutated in IBD-CRC and sporadic CRC included SMAD2/4, PIK3CA, SOX9, FBXW7, and TCF7L2 (Table 2). All three PIK3CA mutations occurred at hotspots previously associated with CRC and other cancers. Likely oncogenic driver gene mutations in BRAF, CTNNB1, and CREBBP were found in one IBD-CRC case each (CHASM scores < 0.2).
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The mutation spectrum of TP53 is of particular interest in light of its prevalence and documented association with environmental exposures77. As expected, the majority of TP53 mutations (14/19) observed in IBD-CRC tumors were missense (Supplementary Table 7), and located predominantly in the DNA binding domain of the protein (aa.100–300, Figure 4). However, there were some notable differences in the spectrum of TP53 single substitutions in IBD-associated neoplasia and sporadic CRC. Specifically, no mutations were observed at the hotspot R273, and only one mutation was found at each of the hotspots R248, G245 and R175, in IBD-associated colorectal tumors. Instead, three of the 19 tumors harbored the less common R282W mutation, and two tumors each had mutations in R158, H179, or R342, which are rarely found in sporadic CRC (Supplementary Table 7). TP53 alterations were found in eight UC- and nine CD-associated tumors. In IBD-associated colorectal tumors, the predominant substitution was a C:G>T:A transition at CpG dinucleotides (n=10, 52.6%). This type of substitution in TP53 has previously been positively correlated with expression of the enzyme inducible NO synthase (iNOS)78 in colonic tumors, where it has been hypothesized to be readout of inflammation-associated DNA damage. Indeed, colonic iNOS expression is known to be increased in IBD79, 80.
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Copy Number Alterations in IBD-Associated CRCs We identified 15 regions with at least five-fold amplification many of which were recurrent or had clinically-relevant genes within them (Supplementary Table 8). These included 8q24.21, containing the MYC oncogene, and 17q12, involving ERBB2, as previously reported in sporadic CRC60, 81. CDX2, a homeobox transcription factor involved in intestinal development82, was the most commonly amplified gene. Recurrently amplified
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regions containing IRS2, involved in activation of the PI3K pathway and recently identified as a candidate driver of CRC83, or containing Suppressor of Cytokine Signaling 1 (SOCS1), were also identified. The only recurrently deleted region spanned the HLA-DQB1 locus in 6p21, a region previously associated with UC risk84. Interestingly, this alteration was found in 6 patients with UC, 1 patient with CD and 1 patient with indeterminate colitis. Pathways Altered in IBD-CRC The set of mutated genes was enriched for gene ontologies associated with cell communication, cell-cell signaling, and cell adhesion (Supplementary Table 9). These findings are consistent with dysregulated cytokines and inflammatory mediators playing a role in IBD-associated CRC.
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We grouped mutated genes into pathways and functions to better understand the overall consequences of mutations in IBD-CRC and how the pathways and functions altered in IBD-CRC compare to those altered in sporadic CRC. We found recurrent alterations in the p53, RTK, RAS, PI3K, WNT, and TGF-ß pathways, as has been previously described for sporadic CRC60. In addition to these established CRC-associated pathways, genes involved in the Rho GTPase network, cell communication/adhesion, cytokine signaling, and chromatin remodeling were recurrently altered in IBD-CRC.
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WNT signaling pathway alterations involving inactivation of APC or activation of CTNNB1 were found in only 17% (5/30) non-hypermutated IBD-CRC tumors, however, recurrent mutations were found on EP300 and SOX9, also thought to be involved in WNT signaling. Several other genes in this pathway, including FZD8, AXIN1, TCF7L2, NKD2, MAP3K7, NLK, ARID1A, and CREBBP harbored private mutations (Supplementary Figure 4). Even considering these, only 53% of the IBD-CRCs had mutations in the WNT signaling pathway, a much lower proportion than has been reported in sporadic CRC60. To directly evaluate the status of WNT signaling in IBD-CRC, we performed beta-catenin immunohistochemical staining in 17 tumors with available material (Supplementary Figure 5; Supplementary Table 10). Not surprisingly, two tumors with APC inactivating and one with CTNNB1 activating mutations showed nuclear accumulation of beta-catenin. TCF7L2 and SOX9 are also considered part of the WNT canonical pathway and two tumors with inactivating mutations in these genes also showed beta-catenin nuclear accumulation. In addition, two tumors without obvious mutations in genes that are part of the canonical WNT pathway showed beta-catenin nuclear staining, although we can’t exclude large deletions of APC that would be missed by exome sequencing. Thus, despite the lower rate of inactivating APC or activating CTNNB1 mutations, the WNT pathway is still dysregulated in IBD-CRC through activating mutations in other components of the pathway. However, the rate of 41% nuclear accumulation of beta-catenin is still much lower than that in sporadic CRCs. The overall rate of mutation in TGF-ß signaling pathway was similar to that of sporadic CRC60. We found SMAD2/4 mutations in 7 tumors (23%), more commonly than in a previous single gene study85. Infrequent mutations of other genes in this pathway, including solitary mutations of TGFBR2, ACVR1, ACVR2B, and SMAD3, were identified in IBD-CRCs (Supplementary Figure 4). Recurrent alterations in genes involved in receptor-mediated signaling pathways were identified in 60% of IBD-CRCs (Supplementary Figure 4). These
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included mutually exclusive amplification of IRS2 and ERBB2, and mutually exclusive mutations of PIK3CA and KRAS, as well as NRG1 mutations.
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Four recurrently mutated genes, DOCK2, DOCK3, PREX2, and RADIL, belonging to the Rho and Rac GTPase network, were mutated in 30% of IBD-CRCs (9/30). Perusing the list of genes with at least one non-silent somatic mutation, we found mutations in several other activating RhoGEFs, along with inactivating RhoGTPase-activating proteins (RhoGAPs)86. Overall, the Rho and Rac pathway was altered in 50% of cases (Supplementary Figure 4). Moreover, a striking 50% of cases (15/30) presented mutations in chromatin remodeling genes, including recurrent mutations in TRRAP and EP300, and low-frequency mutations in CREBBP, ARID1A, and others (Supplementary Figure 4). Dysregulation of cytokine signaling is characteristic of IBD87. We found recurrent mutually exclusive mutations in IL-16 and amplification of SOCS1 in 7 (24%) of cases (Supplementary Figure 4). Genetic alterations functionally associated with cell-cell communication or adhesion were found in 60% of IBD-CRCs, owing to recurrent alterations in CSMD2/3, DOCK2/3, CTNND2, DSCAML1, PTPRF and RADIL (Supplementary Figure 4). Comparison of genes and pathways altered in UC and CD-associated CRC
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Although both UC and CD patients are at elevated risk of developing CRC compared to the general population, the incidence is higher in patients with extensive colitis88. This may be related to more sustained and pervasive inflammation of the colonic mucosa in UC patients. We looked at the overall involvement of each major pathway identified per tumor and compared how these pathways were differentially altered in UC vs. CD (Figure 5). We found that the Rho and Rac pathway was affected in 10 CRCs from confirmed UC patients but in only 3 CRCs from confirmed CD patients (Fisher’s exact test P = .025). With the caveat of our relatively small sample size, it appears that the Rho and Rac pathway may be preferentially activated in UC patients.
DISCUSSION
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IBD patients experience a higher incidence of CRC than the general population. Studies involving limited numbers of genes have suggested that chronic inflammation-driven CRC possesses unique genetic features. Herein, we have presented a comprehensive characterization of the genomic landscape of IBD-CRC and compared it to that of sporadic colorectal tumors. We identified a number of similarities between IBD-associated and sporadic colorectal tumors, indicating that the development of these tumors involves, for the most part, the same cellular pathways. However, there are striking differences. Mutations in key genes involved in the development and progression of sporadic CRC are uncommonly altered in IBD-CRC, suggesting an underlying difference in mechanism or etiology between IBD-associated and sporadic colorectal tumors. Almost all sporadic colorectal tumors have altered WNT pathway genes, most commonly APC. By contrast, we found a lower rate of APC inactivation, but a higher relative number of inactivating mutations in the SOX9 transcription factor, an intestinal stem cell marker89 that antagonizes WNT/beta-catenin signaling90. It has been previously suggested that WNT signaling can be epigenetically activated in IBD-CRC51, thus bypassing the need for APC Gastroenterology. Author manuscript; available in PMC 2017 April 01.
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mutations. Moreover, the WNT pathway could be activated by inflammation in the absence of APC or CTNNB1 mutations, possibly through NF-kB and STAT355. These non-genetic pathways need to be formally explored experimentally. Additionally, we discovered recurrent mutations in components of the Rho and Rac GTPase network and cytokine signaling that support a role for non-canonical WNT signaling in IBD-associated neoplasia. Guanine nucleotide exchange factors (GEFs) promote activation of the Rho GTPase Rac1, which in the colon mediates the production of reactive oxygen species and activation of NFκB, leading to stimulation of WNT signaling and expansion of the intestinal stem cell pool91. Interestingly, abrogation of Rac1 activity pharmacologically92 or genetically91 in animal models prevents the development of colon cancer, suggesting a potential therapeutic benefit from targeting the Rho and Rac pathway in IBD.
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Similar to APC, the prevalence of KRAS mutations in IBD-CRCs was much lower than in sporadic colorectal tumors, while this was not true for SMAD4 and PIK3CA. Approximately 60% of IBD-CRCs had mutations in TP53, a rate equivalent to that of sporadic colorectal tumors. However, hotspot mutations were different between the IBDCRCs and sporadic colorectal tumors. Prior work has suggested that TP53 mutations are acquired early in the IBD-CRC tumorigenic process whereas in sporadic CRCs they have been observed late in the transition from adenoma to carcinoma93, 94. We observed a TP53 mutation rate matching that of sporadic CRCs potentially arguing against this prior observation. However, our study did not include precancerous lesions; therefore, our data is inconclusive regarding the timing of mutations in IBD-CRC. Future studies that evaluate a larger number IBD-CRCs at various stages of development will be required to help delineate this process better.
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A number of mutations and copy number alterations in targetable oncogenic driver genes with well-established ties to sporadic CRC were also found in IBD-CRC. These included hotspot driver mutations in PIK3CA and BRAF, and amplification of IRS2 and ERBB2.
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The presence of recurrent mutations in interleukin-16 (IL16) is particularly interesting, as this cytokine is known to aggravate immune-mediated and autoimmune inflammatory conditions, including IBD95. Serum levels of IL-16 are higher in CRC patients and IL16 gene polymorphisms are associated with susceptibility to CRC96. Expression of IL-16 is upregulated in the colonic mucosae of IBD patients97, 98, and its neutralization reduces injury and inflammation in an animal model of colitis97. Although predictions regarding the functional effect of substitutions are limited by the lack of reliable 3-D models and computational methods to assess most of the uncommon mutations, the available predictions would suggest that mutations in IL-16 could have damaging consequences (Supplementary Table 5). Further studies are required to experimentally determine their specific functional effect. Complementary to our study, sequencing approaches that comprehensively determine mutations in introns, and long noncoding RNA’s, and molecular studies that evaluate epigenetic silencing of tumor suppressors/activation of oncogenes will be an important next step, which should provide further insight into the molecular changes that promote colorectal tumorigenesis in patients with IBD.
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Our finding of a pattern of base changes at AA dinucleotides resembling that found in esophageal and gastric cancers, both of which are tied to chronic inflammation, provides clues to a putative mutagenic role of inflammation that also merits further scrutiny. In summary, this in-depth analysis of colorectal tumors associated with IBD demonstrates a unique molecular profile and provides clues to the etiology of CRC in IBD patients. Our study also sets the stage for improved early detection of colorectal tumors in IBD patients based on their unique genetic composition, as well as for the tailoring of therapies in this patient population.
Supplementary Material Refer to Web version on PubMed Central for supplementary material.
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Acknowledgments We thank Ms. Laura Nakatsuka and Mr Barrett Goodspeed from the MGH Pathology Department for their assistance in specimen identification and procurement. Grant Support This work was supported by the Intramural Research Program of the National Cancer Institute, NIH, Virginia and D.K. Ludwig Fund for Cancer Research, The Sol Goldman Sequencing Facility at Johns Hopkins, and NIH Grants CA43460 (NP), CA057345 (NP), CA152753 (NP), T32-DK007191-38-S1 (GT), CA62924 (RHH), CA133012 (SJM), and CA190040 (SJM & FMS).
Abbreviations Author Manuscript
CRC
colorectal cancer
FFPE
formalin-fixed paraffin-embedded
GEF
Guanine nucleotide exchange factor
GWAS
genome-wide association studies
MSI
microsatellite instability
MSS
Microsatellite-Stable
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Figure 1.
A. Number of genetic alterations (mutations, small insertions/deletions, focal amplifications) detected through sequencing and copy number analyses in each of 32 IBD-associated tumors. Samples are organized by descending total number of alterations and type of IBD. B. Median number of somatic mutations per tumor in non-hypermutated IBD-Colorectal (this study, red frame) and in other tumors of the GI tract. Horizontal bars indicate the 25 and 75% quartiles. MSI, microsatellite instability; hypermut, hypermutable phenotype; EAC, esophageal adenocarcinoma; MSS, microsatellite stable; ESCC, esophageal squamous cell
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carcinoma. Published data on which this figure is based are provided in Supplementary Table 4.
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Author Manuscript Author Manuscript Author Manuscript Figure 2.
Author Manuscript
A. Mutation sequence context of non-hypermutated IBD-associated tumors. Base substitutions were collated into categories representing the 6 possible base changes (represented by colors in the upper right) and further subdivided into the 16 possible combinations that take into account the identity of nucleotides flanking the mutated base, or “trinucleotide context” (for more detail see Supplementary Figure 3). The fractional breakdown of mutation counts is shown in the pie chart on the upper left. B. Mutation sequence context of non-hypermutated tumors from UC cases. C. Mutation sequence context of non-hypermutated tumors from CD cases.
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Figure 3.
Genes recurrently mutated in IBD-associated CRC and their prevalence in sporadic CRC (TCGA). Each column denotes an individual tumor and each row represents a gene. Depicted are genes with mutation rates > 10 mutations/Mb, colored by the type of coding mutation, and marked by * if present in COSMIC. Right, percent of mutated cases in this study (black bars) and in sporadic CRC from TCGA (gray bars). Genes marked by * show mutation prevalence different from that of CRC (two-tailed Fisher’s exact test with
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Benjamini-Hochberg correction for multiple testing, P < .05). Bottom, recurrently-amplified genes and private hotspot mutations.
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Figure 4.
TP53 mutation spectrum in IBD-associated CRC. A. Mutation signature of 19 IBDassociated tumors with TP53 mutations compared to that of all somatic TP53 mutations in human sporadic CRC obtained from the IARC TP53 database (http://p53.iarc.fr/). B. Distribution of TP53 single nucleotide substitutions in IBD-associated CRC. C. Distribution of TP53 single nucleotide substitutions in sporadic CRC obtained from the IARC TP53 database. D. Distribution of TP53 single nucleotide substitutions in sporadic CRC identified
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by exome sequencing in the TCGA study, obtained from cBioPortal (http:// www.cbioportal.org/).
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Author Manuscript Author Manuscript Figure 5.
Author Manuscript
Signaling pathways affected in CD and UC. Genes recurrently mutated or amplified were assigned to manually curated pathways based on KEGG, Gene Ontology, Pathway Commons, and Ingenuity Pathway Analysis (IPA Ingenuity Systems, http:// www.ingenuity.com). Pathways for discussion were selected based on prior association with CRC or the presence of multiple recurrently mutated genes in the pathway.
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Author Manuscript
Author Manuscript 3 3 4 3 4 4 3 3 3 3 3 4 3 5 3 3 3 3 5 5 3 3 4 3
SMAD2
SOX9
NRG1
TPO
IL16
RIMS2
PIK3CA
RADIL
CTNND2
RRBP1
ZCCHC6
DOCK3
PREX2
APC
DOCK2
PTPRF
DSCAML1
CACNA1D
CSMD3
TRRAP
EP300
MTOR
CSMD2
UBR5
4
SMAD4 4
6
SRRM4
19
KRAS
No. of mutations
TP53
Gene symbol
Gastroenterology. Author manuscript; available in PMC 2017 April 01. 3
4
3
3
4
5
3
3
3
3
4
3
4
3
3
3
3
3
4
4
3
4
3
3
3
4
6
19
No. of mutated samples
10%
13%
10%
10%
13%
17%
10%
10%
10%
10%
13%
10%
13%
10%
10%
10%
10%
10%
13%
13%
10%
13%
10%
10%
10%
13%
20%
63%
Percent of cases altered
8400
10896
7650
7245
11580
11124
6546
6342
5724
5493
8532
4821
6093
4488
4233
3678
3228
3207
4236
3999
2802
2142
1530
1404
1836
1659
570
1182
Gene size (bp)
12
12
13
14
14
15
15
16
17
18
20
21
22
22
24
27
31
31
31
33
36
62
65
71
73
80
351
536
Mut / Mb
Yes
–
Yes
Yes
Yes
Yes
Yes
–
Yes
Yes
Yes
Yes
Yes
–
–
–
–
Yes
Yes
–
Yes
–
–
Yes
–
Yes
Yes
Yes
Mutation in Biologically Relevant Gene
Author Manuscript
Recurrently mutated genes in 30 non-hypermutated IBD-associated tumors
–
–
Yes
Yes
–
–
–
–
–
–
Yes
–
–
–
–
–
–
Yes
–
–
–
–
–
Yes
–
Yes
Yes
Yes
Mutation in Clinically Relevant Gene
Yes
–
–
Yes
Yes
–
Yes
–
–
–
Yes
–
–
–
–
–
–
Yes
–
–
–
Yes
–
–
–
Yes
Yes
Yes
Mutation in COSMIC Cancer Gene Census
Author Manuscript
Table 1 Robles et al. Page 24
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Table 2
Author Manuscript
Recurrently mutated genes in non-hypermutated sporadic CRC and their mutation frequency in IBD-CRC
Author Manuscript
Percent of sporadic CRC cases altereda
Percent of IBD-CRC cases altered
P valueb
APC
81%
13%
0.0001
TP53
60%
63%
NS
KRAS
43%
20%
0.016
PIK3CA
18%
10%
NS
FBXW7
11%
7%
NS
SMAD4
10%
13%
NS
NRAS
9%
0%
NS
TCF7L2
9%
7%
NS
FAM123B
7%
0%
NS
SMAD2
6%
10%
NS
CTNNB1
5%
3%
NS
KIAA1804
4%
0%
NS
ACVR1B
4%
0%
NS
GPC6
4%
3%
NS
SOX9
4%
10%
NS
EDNRB
3%
0%
NS
Gene symbol
a
Sporadic CRC mutation profile derived from TCGA.
b
Based on Fisher’s Exact test
NS, not significant
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