GASTROENTEROLOGY 2012;xx:xxx Field Cancerization in the Intestinal Epithelium of Patients With Crohn’s Ileocolitis SUSAN GALANDIUK,*,‡,§ MANUEL RODRIGUEZ–JUSTO,ʈ ROSEMARY JEFFERY,* ANNA M NICHOLSON,‡ YONG CHENG,*,** DAHMANE OUKRIF,ʈ GEORGE ELIA,¶ SIMON J LEEDHAM,# STUART A C McDONALD,*,‡ NICHOLAS A WRIGHT,*,‡ and TREVOR A GRAHAM* *Histopathology Laboratory, Cancer Research UK London Research Institute, London, England; ‡Centre for Digestive Diseases, Blizard Institute of Cell and Molecular Science, and ¶Centre for Tumour Biology, Institute of Cancer and CR-UK Clinical Centre, Barts and the London School of Medicine and Dentistry, London, England; § Department of Surgery, University of Louisville, Louisville, Kentucky; ʈDepartment of Histopathology, University College London Hospital, London, England; # Molecular and Population Genetics Laboratory, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, England; and **Department of Gastrointestinal Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing, Peoples Republic of China AQ: 5-6 BACKGROUND & AIMS: Tumors that develop in patients with Crohn’s disease tend be multifocal, so field cancerization (the replacement of normal cells with nondysplastic but tumorigenic clones) might contribute to intestinal carcinogenesis We investigated patterns of tumor development from pretumor intestinal cell clones METHODS: We performed genetic analyses of multiple areas of intestine from 10 patients with Crohn’s disease and intestinal neoplasia Two patients had multifocal neoplasia; longitudinal sections were collected from patients Individual crypts were microdissected and genotyped; clonal dependency analysis was used to determine the order and timing of mutations that led to tumor development RESULTS: The same mutations in KRAS, CDKN2A(p16), and TP53 that were observed in neoplasias were also present in nontumor, nondysplastic, and dysplastic epithelium In patients, carcinogenic mutations were detected in nontumor epithelium years before tumors developed The same mutation (TP53 p.R248W) was detected at multiple sites along the entire length of the colon from patient; it was the apparent founder mutation for synchronous tumors and multiple dysplastic areas Disruption of TP53, CDKN2A, and KRAS were all seen as possible initial events in tumorigenesis; the sequence of mutations (the tumor development pathway) differed among lesions CONCLUSIONS: Pretumor clones can grow extensively in the intestinal epithelium of patients with Crohn’s disease Segmental resections for neoplasia in patients with Crohn’s disease might therefore leave residual pretumor disease, and dysplasia might be an unreliable biomarker for cancer risk Characterization of the behavior of pretumor clones might be used to predict the development of intestinal neoplasia Keywords: Inflammatory Bowel Disease; Tumor Progression; Clonal Ordering; Oncogene; CRC L ong-standing Crohn’s disease (CD), an inflammatory bowel disease, is associated with an increased risk of developing intestinal cancer that is between and times greater than that of the healthy population.1– Compared with non–inflammatory bowel disease tumors, synchro- nous and multifocal neoplasia occurs in as many as 30% of patients with CD with neoplastic disease.5–7 This suggests that field cancerization, the widespread replacement of the normal cell population by a histologically nondysplastic mutant clone that is predisposed to tumor development,8 –10 could underlie tumorigenesis in CD The identification and characterization of pretumor mutant fields in patients with CD could provide better predictors of a patient’s risk of neoplasia The widely accepted paradigm for tumorigenesis, the somatic mutation theory, is that tumorigenesis begins with rate-limiting mutations in a key growth control gene, resulting in immediate lesion growth, and then subsequent alteration of other genes drives the evolution of subclones within the tumor, leading eventually to the development of a malignant clone.11,12 However, the identification of field cancerization in the lung13 and colon14,15 and skin16 makes it increasingly clear that tumor development can begin long before any overt tumor growth.17 The dynamics of such pretumor clones remain singularly unknown Determining parameters such as the clone growth rate would permit more precise estimations of a patient’s risk and timescale for neoplastic development, while also revealing the basic biology of stem cell clone growth in the human intestine Given their risk of malignancy, patients with CD are often subjected to routine endoscopic surveillance with concomitant surgery.18 –20 Thus, tissue samples are collected in some patients over many years, providing a fortuitous but uncommon means to study the longitudinal dynamics of pretumor clones in the human intestine The genetic pathways of tumor development in CD have not been conclusively determined; however, the etiologic similarities between CD and ulcerative colitis (UC) suggest that tumorigenesis in the diseases may share genetic pathways UC-associated neoplasia frequently shows TP53 or KRAS mutations,14,21–23 whereas inactivating mutations of the adenomatous polyposis coli (APC) Abbreviations used in this paper: CD, Crohn’s disease; LOH, loss of heterozygosity; PCR, polymerase chain reaction; UC, ulcerative colitis © 2012 by the AGA Institute 0016-5085/$36.00 doi:10.1053/j.gastro.2011.12.004 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 BASIC AND TRANSLATIONAL AT 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 gene, found in most sporadic colorectal tumors,24 are rarely observed.14,22 Loss of heterozygosity (LOH) of chromosome arm 18q, possibly targeting the SMAD4 gene, is also relatively frequent in UC cancer.21,25 Here, we investigate pretumor clonal dynamics in the inflamed human CD bowel Inferring clonal dynamics in the human bowel is technically challenging, because invasive labeling studies are impractical Instead, we used clonal ordering techniques that exploit the relative spatial location of mutations to infer the order in which they were acquired.26,27 Rare point mutations mark clones, because cells are very unlikely to bear the same point mutation unless they share a clonal origin Longitudinally collected tissue samples allow the extent and rate of clonal expansion to be determined, indicating the relative fitness of each clone.28 Patients and Methods See Supplementary Materials and Methods for full methods Patient and Clinical Material T1 BASIC AND TRANSLATIONAL AT 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 GALANDIUK ET AL Paraffin-embedded biopsy and resection specimens were obtained from 10 patients with CD-associated intestinal neoplasia (Table 1) Specimens from multiple areas of intestine were available from each patient; patients had longitudinally collected samples, and patients had multifocal neoplasia The case of patient is particularly enlightening because this patient had tissue collected over an 8-year period (1996 –2004), during which he developed multifocal and metachronous tumors Mutation Screening of Cancers Initially, DNA was needle macrodissected from each neoplastic lesion (surgical resection specimens) and screened for somatic mutations using polymerase chain reaction (PCR) sequencing at loci frequently mutated in inflammatory bowel disease cancers: TP53 exon 5–9, KRAS exon (codons 12–13), and CDKN2A (p16INK4a) exon Primer details are listed in Supplementary Table Laser Capture Microdissection AQ: AQ: Mutations identified in the screening phase were then investigated on a crypt-by-crypt basis in multiple tissue specimens from each patient A total of 450 crypts were microdissected from serial sections using a PALM microdissection system (Zeiss, Munich, Germany); in poorly differentiated tumors, small areas of epithelial cells were microdissected Pathology was assessed using a serial H&E-stained slide Following DNA extraction by digestion in PicoPure proteinase K buffer (Applied Biosystems, Warrington, United Kingdom), DNA lysates were PCR sequenced Tubes containing digestion buffer but no microdissection material were included as negative controls LOH Analysis AQ: LOH analysis was performed on individual crypt lysates at up to 19 informative markers close to the FHIT, APC, CDKN2A, SMAD4, and TP53 genes, respectively, using a multiplexed microsatellite PCR kit (Qiagen, Crawley, United Kingdom) LOH was then considered present if the area under one allelic peak was more than twice that of the other, after normalizing peak areas relative to constitutional DNA Multiple infor- GASTROENTEROLOGY Vol xx, No x mative markers located close to APC, TP53, and CDKN2A were available for some patients; in these cases, LOH was called if it was shown by at least of the available markers Microsatellite instability was assessed using multiplexed assay for the BAT-25 and BAT-26 mononucleotide repeats29; the presence of microsatellite instability in a sample precluded reliable LOH analysis Primer details are listed in Supplementary Table Image Cytometry Areas of approximately mm2 of epithelium were needle dissected from waxed 40-m sections, using a serial H&E section as a guide Image cytometry was performed using the Fairfield DNA Ploidy System (Fairfield Imaging, Kent, United Kingdom) as previously described.14 Immunohistochemistry Immunohistochemistry was performed for p53 (Dako, Ely, United Kingdom), -catenin (Transduction Labs, Oxford, United Kingdom), and lysozyme (Dako) using standard protocols Results Somatic mutations in TP53, CDKN2A, or KRAS were detected in neoplastic tissue from of 10 patients (Table 1) Individual histologically normal, inflamed, or dysplastic crypts, cancer glands, or small areas of poorly differentiated tumor (ϳ500 cells) were then microdissected from multiple tissue samples from each patient with detected mutations and genotyped for the somatic mutation(s) detected in the patient’s neoplasia (Supplementary Table 3) Clonality of Tumor and Nontumor Tissue Individual crypts from nontumor tissue were examined in the of 10 patients who had a detected mutation in their neoplasia In of of these patients, the mutation present within the tumor could also be detected in nontumor tissue (Figure and Supplementary Table 3), suggesting in each case a clonal relationship between the tumor and nontumor mucosa Within nontumor tissue, the mutations found in the cancer were frequently present in both dysplastic and nondysplastic crypts, although always in areas of active disease (Figure and Supplementary Table 3) Patients and had cancer in the small intestine, whereas patients 1, 5, and had colon cancer, indicating that pretumor clone growth occurred in both intestinal compartments The remaining patients (patients and 7) had detected mutations only within the cancer and dysplasiaassociated lesion or mass, respectively Although an undetected pretumor clone may have preceded tumor growth, it is conceivable that these neoplasia followed a sporadic pattern of tumorigenesis Detection of Mutant Clones Many Years Before Cancer Growth and at Sites Distinct From the Cancer Longitudinally collected samples, available from informative patients (1 and 5), provided a means to study the dynamic behavior of putative pretumor clones (Figure 58 59 60 61 62 63 64 65 66 67 68 AQ: 10 69 70 71 72 AQ: 11 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 F1 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 Month 2012 Table Patient Details Patient Age (y) CD diagnosed Age (y) neoplasm diagnosed Family history of inflammatory bowel disease Family history of colorectal cancer 65 73 No No Location of CD Pancolonic and terminal ileum Immunomodulator use No No of neoplasms Location of neoplasm Sigmoid Sigmoid Rectum 39 39 No Yes Ileocolic No Sigmoid 44 70 No No Small bowel and ileocolic Yes, azathioprine Jejunum 29 30 Yes Yes Ileocolic No Right colon 37 65 No No Yes, azathioprine Perineal wound in fistula tract 37 52 Yes No Pancolonic terminal ileum, jejunum perianal Colonic No Rectal 71 86 No No Colonic No 10 32 Yes Yes Ileocolic No Transverse colon Ileum 45 46 No No Ileocolic perianal No Right colon 10 78 79 No No Ileocolic No Right colon TP53 Moderately differentiated adenocarcinoma High-grade dysplasia Poorly differentiated adenocarcinoma TP53 Moderately differentiated adenocarcinoma Moderately differentiated colloid adenocarcinoma Moderately differentiated adenocarcinoma Well-differentiated mucinous adenocarcinoma TP53 Moderately differentiated adenocarcinoma Adenomas (DALM) TP53 Kras Exon c.817CϾT R273C None Poorly differentiated mucinous adenocarcinoma Poorly differentiated adenocarcinoma Mucinous moderately differentiated adenocarcinoma None Exon c.731GϾA G244D Exon c.742CϾT R248W Exon c.742CϾT R248W Exon c.637CϾT R213* None Kras p16 Tissues examined over time None None 1996 1998 2000 2001 2003 2004 None None No c.35GϾA G12D c.442CϾA A148T No None None No c.37GϾA G13D c.238CϾT R80* None None No None None c.35GϾA G12D None None 2009 2010 No None None None None No None None None None No Kras p16 TP53 TP53 Kras p16 Exon c.637CϾT R213* Exon c.733GϾA G245S 2004 2006 2008 DALM, dysplasia-associated lesion or mass as adenoma was in area of colitis FIELD CANCERIZATION IN CROHN’S DISEASE Type of neoplasm(s) Mutant genes detected 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 BASIC AND TRANSLATIONAL AT BASIC AND TRANSLATIONAL AT 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 C 203 O 204 L O 205 R 206 AQ: 20715 208 209 210 211 212 213 214 215 F2,AQ: 12 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 GALANDIUK ET AL GASTROENTEROLOGY Vol xx, No x Figure Mutations present in nontumor tissue (A) (i) H&E stain (original magnification 100ϫ) of nondysplastic (hyperplastic) mucosa showing crypt distortion with increased inflammatory cells and intraepithelial neutrophils in the transverse colon in patient in 2004 and (ii) serial methylene green–stained PALM laser capture slide showing microdissected crypt (iii) Sequencing shows the crypt contains a TP53 c.742CϾT mutation (B) (i) H&E stain (original magnification 100ϫ) of nondysplastic (hyperplastic) mucosa with a marked increase in the inflammatory cells in the lamina propria and cryptitis in the resection margin of the sigmoid cancer resected from patient in 2000 with (ii) serial PALM slide showing microdissection (iii) Sequencing shows the crypt contains a TP53 c.775GϾT mutation (C) p53 immunohistochemistry on a nondysplastic colon resection specimen from patient (original magnification 100ϫ) (i) Patches of crypts with nuclear accumulation of p53 protein were observed, frequently demarked by odd p53-negative or low-expressing crypts (ii and iii) (arrows) (D) (i) H&E stain (original magnification 40ϫ) of inflammatory atypia within cells in a perianal fistula from patient four years before tumor growth (ii) Laser capture slide (original magnification 10ϫ) (iii) TP53, CDKN2A, and KRAS mutations in each crypt and Supplementary Figure 1) Patient developed an adenocarcinoma arising within a perineal proctectomy scar that was resected in 2008 Both the cancer and the nearby nondysplastic crypts in the inflamed resection margin contained KRAS c.37GϾA, TP53 c.733GϾA, and CDKN2A mutations, and the same mutations could also be detected in a proctectomy specimen collected years earlier (Supplementary Table 3) However, a nearby perianal fistula tract in the earlier proctectomy specimen showed the same KRAS c.37GϾA mutation, a different TP53 mutation, and no CDKN2A mutation, indicating that KRAS c.37GϾA was the first pretumor mutation This second TP53 mutation was not detected in later samples, suggesting the clone had died out (Supplementary Figure 1) Interestingly, a small bowel biopsy specimen from 2004 showed a different KRAS c.35GϾA mu- tant clone in nondysplastic crypts; however, a subsequent biopsy specimen from the same area in 2006 did not contain this clone, suggesting this second KRAS clone had undergone only limited clonal expansion or died out An extensive tissue archive was available from patient (Figure and Supplementary Table 3) The sigmoid adenocarcinoma resected from this patient in 2000 contained a TP53 c.731GϾA mutation throughout (Supplementary Figure 2), which was also present in morphologically nondysplastic crypts in the resection margin Crypts from earlier rectal and sigmoid biopsy specimens (1996 and 1998) contained no detected TP53 mutations, suggesting that the TP53 c.731GϾA mutation occurred in the years before cancer growth A follow-up biopsy around the anastomosis area did not contain the c.731GϾA mutation, suggesting the resection successfully removed the mutant clone 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 Month 2012 C O L O R Figure Longitudinal analysis showing clone spread in patient Tissue collected before 2000 contained no widespread detected genetic abnormalities A c.731GϾA TP53 mutation was first detected throughout a sigmoid cancer in 2000 and within the resection margins, but not in later samples, suggesting the mutant arose in the sigmoid after the biopsy in 1998 and was completely removed by the cancer resection A second c.775GϾT TP53 was also found in the resection margin of the sigmoid cancer This second clone had spread to the transverse colon by 2004 A third TP53 c.742CϾT mutation was first detected in sigmoid and rectal biopsy specimens from 2001 By 2004, the mutant TP53 c.742CϾT clone was present in every major segment of the colon and also in the terminal ileum and in multifocal neoplasia Shapes indicate the predominant pathological diagnosis at the site, and color indicates the predominant TP53 genotype (see key) A second TP53 mutant, c.775GϾT, was detected in the inflamed but nondysplastic resection margin of the sigmoid cancer in 2000 The same mutation was later detected in the transverse colon mucosa resected in 2004 A third TP53 c.742CϾT mutation was also detected in this patient, in multiple neoplastic lesions, and at multiple sites spanning the length of the colon (Figure 2) The mutation was first identified in crypts from sigmoid and rectum collected in 2001 and was not present in earlier samples, making it likely the mutation was first acquired in this area around this time The c.742CϾT mutation was again detected in sigmoid crypts collected in 2003 but not in a simultaneous rectal biopsy specimen By 2004, the c.742CϾT mutation was detected at sites along the entire length of the colon: a rectal cancer (Supplementary Figure 3), a transverse colon hyperplastic mucosa (Figure 1), a right colon adenoma, and in the nondysplastic but 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 BASIC AND TRANSLATIONAL AT 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 FIELD CANCERIZATION IN CROHN’S DISEASE inflamed mucosa surrounding each of these lesions This mutation was a founding mutation for areas of dysplasia, an adenoma, and a cancer Immunohistochemical analysis of the patient’s colectomy specimen in 2004 revealed patchy and infrequent p53 overexpression along the colon length (Figure 1), suggesting that p53 mutant clones were present only in a minority of crypts Noninflamed areas of terminal ileum were available from patients and Crypts microdissected from these nondiseased areas did not contain the mutations found in these patients’ cancer and inflamed mucosa (Supplementary Figure 4) Crypts on Either Side of the Ileal-Cecal Valve Have a Common Somatic Mutation Individual crypts from inflamed terminal ileum tissue from patient collected in 2004 contained the same BASIC AND TRANSLATIONAL AT 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 GALANDIUK ET AL GASTROENTEROLOGY Vol xx, No x C O L O R Figure Presence of the same TP53 mutation in functional small intestinal and colon crypts (A) H&E section showing inflamed terminal ileum mucosa with crypt/villous distortion and an increase in inflammatory cells and intraepithelial neutrophils (B) Serial section stained for lysozyme indicating the presence of functional Paneth cells at the small intestinal crypt base (C) Serial laser capture slide stained with methylene green showing separately microdissected crypt and villus Genotyping revealed the same TP53 c.742CϾT was present in both the crypt and villus This same mutation was found in colon tissue collected at the same and previous times Original magnification 100ϫ for all micrographs F3 TP53 c.742CϾT mutation present in this patient’s colon Separate microdissection of crypts and villi showed that the mutation was also present on the villus fed by a mutant crypt (Figure 3) Immunohistochemistry for lysozyme staining highlighted the presence of Paneth cells within mutated crypts (Figure 3) Interchangeable Order of Mutations in Tumorigenesis F4 The order of mutations in tumorigenesis differed between tumors Mutation order was inferred from the relative spatial localization of mutants; genetic heterogeneity was a hallmark of most tumors In all polypoid lesions detected in patient 1, a TP53 point mutation was present throughout the lesion and within the resection margins, suggesting TP53 was the founder mutation for each lesion Tumor development involved sequential 17p, 9p and 18q LOH, and aneuploidy (Figure and Supplementary Figure 3) The sigmoid cancer resected in patient in 2000 also showed widespread 5q LOH and nuclear -catenin (Supplementary Figure 2), implicating APC in early tumorigenesis of this lesion Biallelic mutation of TP53, presumed on the detection of both a TP53 point mutation and LOH, was not requisite for tumor growth; the ascending colon adenoma in patient in 2004 had a TP53 point mutation throughout the adenoma but only a subclone had 17p LOH; furthermore, biallelic TP53 mutations were infrequently observed in nondysplastic crypts in this patient (Supplementary Table 3) Similarly, a putative biallelic TP53 mutation was observed in the dysplastic margins of the cancers from both patients and The presence of a CDKN2A mutation in both tumor and nondysplastic tissue in patient implicates CDKN2A disruption in the initiation of this patient’s tumor; tumor progression involved a subsequent KRAS mutation and the development of microsatellite instability (Supplementary Figure 4) The initial mutation in patient was in KRAS, with later CDKN2A and TP53 mutations implicated in subsequent tumor development Discussion The genetic and histologic mechanisms driving the development of CD-associated cancers have not been conclusively determined The data presented herein are strong evidence that field cancerization, the replacement of the normal epithelium with a protumorigenic clone,8,9 before any dysplastic histologic change contributes to carcinogenesis in patients with CD In of informative patients with neoplasia, the same point mutation in KRAS, CDKN2A, or TP53 could be detected within the tumor, neighboring resection margins that showed active disease, and even in more distant diseased areas, strongly suggesting that this mutant tissue was clonal in origin In patient, a TP53 c.742CϾT mutation was first detected focally in the sigmoid colon and then years later at sites along the entire colon length These data suggest 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 FIELD CANCERIZATION IN CROHN’S DISEASE 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 BASIC AND TRANSLATIONAL AT Month 2012 C O L O R Figure Phylogenetic trees of neoplasia development (A) Phylogenetic tree of sigmoid cancer resected in patient in 2000 Tumor growth was preceded by the acquisition of a TP53 c.731GϾA mutation, because this mutation was found in the cancer and its resection margin A clone with 5q LOH was dominant in the cancer, and a single crypt showed 9p and 18q LOH but no 5q LOH Numerous small subclones with distinct patterns of 9p, 18q, and 17p LOH were found within the 5q subclone This frequent LOH could be attributable to the cancer containing an aneuploid clone, which was not tested for in this cancer (B) Phylogenetic tree of the ascending colon adenoma resected in patient in 2004 The cancer developed from the preneoplastic clone distinguished by the TP53 c.742CϾT mutation, and subsequent carcinogenesis involved the sequential acquisition of 17p, 9p, and then 18q LOH Unlike the previous cancer in patient 1, no 5q LOH was detected in the lesion in 2004 (C–G) Phylogenetic trees for tumors from patients 1– Most lesions were initiated by a TP53 mutant clone, with the exceptions of patient 3, where the putative founder clone was a CDKN2A (p16) mutant, and patient 5, where the founder clone was a KRAS mutant The inferred sequence of subsequent mutations and LOH events was different in every lesion Colors indicate genotypes (see key), and line thicknesses are indicative of relative clone abundance that the mutant clone had arisen in the left colon and appeared to have spread both proximally to the right colon and distally to the rectum within years (Figure 2), suggesting pretumor growth can occur at a substantial scale and rate This conclusion rests on the strength of the TP53 c.742CϾT point mutation to uniquely identify a clonal population of cells in this patient If this particular mutation occurred at high frequency, it is conceivable that unrelated cells could independently develop the mutation; if this were true, then the data suggest pretumor clone growth of more restricted spatial extent The International Agency for Research on Cancer p53 mutation database30 suggests that the c.742CϾT mutation comprises approximately 7% of the total detected somatic TP53 mutations in the human intestine Given crypts that both have a TP53 mutation, the odds that crypts will both have the c.742CϾT mutation are small (approximately 200:1) This calculation assumes that the distribution of TP53 mutations selected in the CD bowel mirrors that reported in the International Agency for Research on Cancer database for the intestine at large However, given that other gastrointestinal cancers arising in an inflammatory environment show a broad range of TP53 mutations, this is likely a reasonable assumption.14,31–33 The notion that the TP53 c.742CϾT mutation marks a single clone is further supported by LOH data; both the rectal BASIC AND TRANSLATIONAL AT 406 407 408 409 410 411 412 AQ: 13 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 GALANDIUK ET AL and ascending colon lesions in patient 1, which were clonal for the TP53 c.742CϾT point mutation, showed loss of the same alleles of markers on chromosome arms 9p, 18q, and 17p Because the same allele of 17p was consistently lost around the entire colon, this suggests that the universally retained allele bore the c.742CϾT point mutation The odds of finding cells, which both have LOH at these loci and have lost the same alleles, are 1:8; the odds of mutated cells both bearing these same patterns of LOH and the c.742CϾT point mutation are at most 1:1600 This probability must also be considered in light of the presumably very small likelihood of a crypt acquiring a fixed genetic lesion at all Therefore, it is likely that the widely dispersed TP53 c.742CϾT mutant cells, frequently marked by an additional genetic lesions, in patient comprised a single clone Furthermore, evidence of long-range clone spread in this patient is clearer still when the TP53 c.775GϾT mutant is considered This mutation is reported to be relatively rare, comprising approximately 0.1% of the reported somatic TP53 variants in the International Agency for Research on Cancer database, and so the odds of this mutant arising more than once in the bowel are minimal The TP53 c.775GϾT mutation was detected in the descending colon at baseline and the transverse colon years later, indicating extensive clone migration during this time How does a mutant clone grow in the CD intestine? Crypt fission is the primary mechanism of clonal expansion in the intestinal epithelium.34,35 Although fission is rare in the normal intestine, patients with colitis have an elevated fission rate,36 and tumorigenic mutations may further increase the crypt division rate Chronic inflammation, resulting in cycles of crypt atrophy and mucosal healing by crypt fission, likely provides a major growth stimulus; indeed, mutant clones were only identified in areas of active disease Despite this, it is unlikely that a mutant clone could sustain exponential growth,37 raising the possibility that extensive clonal expansion may occur through a noncanonical mechanism such as stem cell migration between crypts, perhaps analogous to the migration between niches observed in the Drosophila ovary.38 p53 and p16 mutants were observed to have undergone extensive clonal expansion in the colitic bowel, indicating that these mutated clones have a selective advantage in the CD intestine Mutations that impair the ability of p53 and p16 to regulate the apoptosis and senescence responses presumably provide a survival advantage, because such mutant clones are likely more able to withstand the inflammatory stress of the colitic bowel Specific clone fitness may be determined by the mutation type; indeed, the c.742CϾT, p.R248W mutant, which underwent the most extensive clonal expansion, is associated with severe down-regulation of p53 activity.30,39 Differential effects of each mutation may also regulate the ability of a mutant cell to compete with wild-type or other mutant cells for a place in the bowel.40 Further selective advantage for TP53 mutants may be due to their tolerance of genetic instabil- GASTROENTEROLOGY Vol xx, No x ity,41,42 seemingly an early event in colitis-associated cancers.43 Supporting this hypothesis, an aneuploid population was observed in transverse colon nondysplastic but p53mutant tissue in patient (Supplementary Table and Supplementary Figure 5) Notably, mutations were only detected in diseased tissue; consequently, we would suggest that there is interdependence between the persistence of mutant clones and inflammatory disease This relationship gives further credence to the suggestion that tumor suppressor gene mutations could confer a survival advantage in the inflamed bowel; indeed, of the patients who had identified field cancerization were also recorded as having long-standing active CD Further, it is tempting to speculate that persistent inflammation may be necessary for significant clone growth Fission rates are markedly increased in inflamed mucosa,36 and potentially this is the direct cause of clone spread Future analyses of isolated “skip lesions” and their histologically normal margins, and also of pathology archives that fully catalog nondiseased bowel, will provide a means to fully investigate any relation between active disease and clone spread Our data are insufficient to investigate any potential relationship between mucosal injury from an endoscopic biopsy and clone growth In one patient, the same TP53 mutation was detected in crypts on both sides of the ileal-cecal valve These data therefore suggest that a colon-derived cell can cross into the small intestine and form functional crypt-villus units The strength of this conclusion depends on the reliability of the TP53 mutation as a clone marker; its merits have been discussed previously There are a number of possible mechanisms for this apparent cell migration First, an impaired ileocecal valve, perhaps compromised by inflammation, could permit retrograde clone expansion Second, the relatively common fissures in patients with CD44 could provide a migratory route for a cryptogenic cell Third, it is conceivable that endoscopy could seed colonderived cryptogenic cells into the small intestine; denuded epithelium in areas of active disease may provide a fertile ground for migrating cells Once in the small intestine, mesenchymal signaling may then induce the progeny of the colon-derived stem cell(s) to follow a small intestinal pattern of differentiation.45,46 The clinical implications of our findings could be considerable First, we have shown that tumorigenic mutations may be present in large sections of morphologically nondysplastic mucosa This questions the adequacy of dysplasia as a biomarker for neoplasia risk, because nondysplastic crypts can carry what may prove to be a biologically significant mutation burden Molecular profiling of the nondysplastic epithelium could potentially better identify patients at risk for neoplasia, with the detection of particular mutants predicting enhanced risk A controlled study comparing the mutant-clone burden and dynamics between patients with CD cancer and patients without cancer over time is required Second, it questions the adequacy of performing endoscopic or limited resections for colonic neoplasms in patients with CD, but close 406 407 408 409 AQ: 14 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 surveillance in patients with long-standing Crohn’s colitis is still needed until more is understood about the process It is conceivable that patients who have developed a prolific mutant clone may be best treated by colectomy rather than limited resection, whereas a less prolific clone could be dealt with by localized treatment, although an assessment of both the efficacy and cost-effectiveness of these suggestions would need to be performed Definite conclusions here are prohibited by the absence of data from patients who never acquired cancers; this is a limitation of our study The remarkable motility of pretumor clones may be a hallmark of other inflammation-associated cancers such as hepatocellular carcinoma, cholangiocarcinoma, and Barrett’s-associated adenocarcinoma, highlighting the need for further investigation into the dynamics of pretumor clones to understand patterns of disease occurrence We have shown frequent and occasionally very extensive field cancerization in the chronically inflamed bowel of a few patients with CD These data provide a precedent for further study of pretumor clones as biomarkers in inflammatory diseases Our observation of nondysplastic crypts harboring tumorigenic clones questions the utility of dysplasia as the sole biomarker of neoplastic risk Foremost, we have highlighted that pretumor clonal dynamics can contribute to patterns of tumor occurrence Supplementary Material Note: To access the supplementary material accompanying this article, visit the online version of Gastroenterology at www.gastrojournal.org, and at doi: 10.1053/j.gastro.2011.12.004 References Laukoetter MG, Mennigen R, Hannig CM, et al Intestinal cancer risk in Crohn’s disease: a meta-analysis J Gastrointest Surg 2011;15:576 –583 Hemminki K, Li X, Sundquist J, et al Cancer risks in Crohn disease patients Ann Oncol 2009;20:574 –580 von Roon AC, Reese G, Teare J, et al The risk of cancer in patients with Crohn’s disease Dis Colon Rectum 2007;50:839 – 855 Canavan C, Abrams KR, Mayberry J Meta-analysis: colorectal and small bowel cancer risk in patients with Crohn’s disease Aliment Pharmacol Ther 2006;23:1097–1104 Gyde SN, Prior P, Macartney JC, et al Malignancy in Crohn’s disease Gut 1980;21:1024 –1029 Connell WR, Sheffield JP, Kamm MA, et al Lower gastrointestinal malignancy in Crohn’s disease Gut 1994;35:347–352 Ribeiro MB, Greenstein AJ, Sachar DB, et al Colorectal adenocarcinoma in Crohn’s disease Ann Surg 1996;223:186 –193 Slaughter DP, Southwick HW, Smejkal W Field cancerization in oral stratified squamous epithelium; clinical implications of multicentric origin Cancer 1953;6:963–968 Braakhuis BJM, Tabor MP, Kummer JA, et al A genetic explanation of Slaughter’s concept of field cancerization: evidence and clinical implications Cancer Res 2003;63:1727–1730 10 Ushijima T Epigenetic field for cancerization J Biochem Mol Biol 2007;40:142–150 11 McCombs R A hypothesis on the causation of cancer Science 1930;72:423– 424 12 Nowell PC The clonal evolution of tumor cell populations Science 1976;194:23–28 FIELD CANCERIZATION IN CROHN’S DISEASE 13 Franklin WA, Gazdar AF, Haney J, et al Widely dispersed p53 mutation in respiratory epithelium A novel mechanism for field carcinogenesis J Clin Invest 1997;100:2133–2137 14 Leedham SJ, Graham TA, Oukrif D, et al Clonality, founder mutations, and field cancerization in human ulcerative colitis-associated neoplasia Gastroenterology 2009;136:542–550.e6 15 Nosho K, Kure S, Irahara N, et al A prospective cohort study shows unique epigenetic, genetic, and prognostic features of synchronous colorectal cancers Gastroenterology 2009;137: 1609 –1620 e1–3 16 Hafner C, Toll A, Fernandez-Casado A, et al Multiple oncogenic mutations and clonal relationship in spatially distinct benign human epidermal tumors Proc Natl Acad Sci U S A 2010;107: 20780 –20785 17 Chung GT, Sundaresan V, Hasleton P, et al Clonal evolution of lung tumors Cancer Res 1996;56:1609 –1614 18 Farraye FA, Odze RD, Eaden J, et al AGA medical position statement on the diagnosis and management of colorectal neoplasia in inflammatory bowel disease Gastroenterology 2010;138:738 – 745 19 Cairns SR, Scholefield JH, Steele RJ, et al Guidelines for colorectal cancer screening and surveillance in moderate and high risk groups (update from 2002) Gut 2010;59:666 – 689 20 Ullman T, Odze R, Farraye FA Diagnosis and management of dysplasia in patients with ulcerative colitis and Crohn’s disease of the colon Inflamm Bowel Dis 2009;15:630 – 638 21 Kern SE, Redston M, Seymour AB, et al Molecular genetic profiles of colitis-associated neoplasms Gastroenterology 1994;107: 420 – 428 22 Rashid A, Hamilton SR Genetic alterations in sporadic and Crohn’s-associated adenocarcinomas of the small intestine Gastroenterology 1997;113:127–135 23 Hussain SP, Amstad P, Raja K, et al Increased p53 mutation load in noncancerous colon tissue from ulcerative colitis: a cancerprone chronic inflammatory disease Cancer Res 2000;60:3333– 3337 24 Rowan AJ, Lamlum H, Ilyas M, et al APC mutations in sporadic colorectal tumors: A mutational “hotspot” and interdependence of the “two hits.” Proc Natl Acad Sci U S A 2000;97:3352–3357 25 Hoque AT, Hahn SA, Schutte M, et al DPC4 gene mutation in colitis associated neoplasia Gut 1997;40:120 –122 26 Graham TA, Wright NA Investigating the fixation and spread of mutations in the gastrointestinal epithelium Future Oncol 2008; 4:825– 839 27 Thirlwell C, Will OCC, Domingo E, et al Clonality assessment and clonal ordering of individual neoplastic crypts shows polyclonality of colorectal adenomas Gastroenterology 2010;138:1441– 1454, 1454.e1–7 28 Maley CC, Galipeau PC, Li X, et al Selectively advantageous mutations and hitchhikers in neoplasms: p16 lesions are selected in Barrett’s esophagus Cancer Res 2004;64:3414 –3427 29 Stone JG, Tomlinson IP, Houlston RS Optimising methods for determining RER status in colorectal cancers Cancer Lett 2000; 149:15–20 30 Petitjean A, Mathe E, Kato S, et al Impact of mutant p53 functional properties on TP53 mutation patterns and tumor phenotype: lessons from recent developments in the IARC TP53 database Hum Mutat 2007;28:622– 629 31 Prevo LJ, Sanchez CA, Galipeau PC, et al p53-mutant clones and field effects in Barrett’s esophagus Cancer Res 1999;59:4784 – 4787 32 Flejou JF, Gratio V, Muzeau F, et al p53 abnormalities in adenocarcinoma of the gastric cardia and antrum Mol Pathol 1999;52: 263–268 33 Yoshida T, Mikami T, Mitomi H, et al Diverse p53 alterations in ulcerative colitis-associated low-grade dysplasia: full-length gene sequencing in microdissected single crypts J Pathol 2003;199: 166 –175 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 BASIC AND TRANSLATIONAL AT Month 2012 10 BASIC AND TRANSLATIONAL AT 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 GALANDIUK ET AL 34 Greaves LC, Preston SL, Tadrous PJ, et al Mitochondrial DNA mutations are established in human colonic stem cells, and mutated clones expand by crypt fission Proc Natl Acad Sci U S A 2006;103:714 –719 35 Gutierrez-Gonzalez L, Deheragoda M, Elia G, et al Analysis of the clonal architecture of the human small intestinal epithelium establishes a common stem cell for all lineages and reveals a mechanism for the fixation and spread of mutations J Pathol 2009;217:489 – 496 36 Cheng H, Bjerknes M, Amar J, et al Crypt production in normal and diseased human colonic epithelium Anat Rec 1986;216:44 – 48 37 Chao DL, Eck JT, Brash DE, et al Preneoplastic lesion growth driven by the death of adjacent normal stem cells Proc Natl Acad Sci U S A 2008;105:15034 –15039 38 Nystul T, Spradling A An epithelial niche in the Drosophila ovary undergoes long-range stem cell replacement Cell Stem Cell 2007;1:277–285 39 Kato S, Han S-Y, Liu W, et al Understanding the function-structure and function-mutation relationships of p53 tumor suppressor protein by high-resolution missense mutation analysis Proc Natl Acad Sci U S A 2003;100:8424 – 8429 40 Moreno E Is cell competition relevant to cancer? Nat Rev Cancer 2008;8:141–147 41 Brentnall TA, Crispin DA, Rabinovitch PS, et al Mutations in the p53 gene: an early marker of neoplastic progression in ulcerative colitis Gastroenterology 1994;107:369 –378 42 Burmer GC, Rabinovitch PS, Haggitt RC, et al Neoplastic progression in ulcerative colitis: histology, DNA content, and loss of a p53 allele Gastroenterology 1992;103:1602–1610 43 Chen R, Rabinovitch PS, Crispin DA, et al DNA fingerprinting abnormalities can distinguish ulcerative colitis patients with dysplasia and cancer from those who are dysplasia/cancer-free Am J Pathol 2003;162:665– 672 44 Williams DR, Coller JA, Corman ML, et al Anal complications in Crohn’s disease Dis Colon Rectum 1981;24:22–24 GASTROENTEROLOGY Vol xx, No x 45 Kedinger M, Lefebvre O, Duluc I, et al Cellular and molecular partners involved in gut morphogenesis and differentiation Philos Trans R Soc Lond B Biol Sci 1998;353:847– 856 46 Benahmed F, Gross I, Gaunt SJ, et al Multiple regulatory regions control the complex expression pattern of the mouse Cdx2 homeobox gene Gastroenterology 2008;135:1238 –1247, 1247 e1–3 Received July 20, 2011 Accepted December 3, 2012 Reprint requests Address requests for reprints to: Susan Galandiuk, e-mail: s0gala01@louisville.edu; fax: (502) 852-8915; or Trevor A Graham, e-mail: trevor.graham@ucsfmedctr.org AQ: AQ: Acknowledgments Trevor A Graham’s present address is: Center for Evolution and Cancer, University of California, San Francisco, 2340 Sutter Street, Box 1351, San Francisco, California 94143 The authors thank members of the Equipment Park and Experimental Histopathology Laboratories at the Cancer Research UK London Research Institute for technical assistance Conflicts of interest The authors disclose no conflicts AQ: Funding AQ: S.G received funding from the Price Institute of Surgical Research, University of Louisville, and Sarah Shallenberger Brown and support in part from National Institutes of Health/National Institute of Environmental Health Sciences grant 1P30ES014443-01A1 T.A.G., R.J., S.J.L., and N.A.W were supported by Cancer Research UK M.R.-J is supported by UCLH/UCL Comprehensive Biomedical Research Centre T.A.G and M.R.-J received funding for this study from the University College London Hospitals Charities - Fast Track Grant 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 Month 2012 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 AQ: 16 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 AQ: 17 629 630 631 632 633 634 635 Supplementary Materials and Methods Patient and Clinical Material Following institutional review board approval and written informed consent, unique paraffin-embedded biopsy and resection specimens were obtained from 10 patients with Crohn’s colitis with neoplasia Specimens consisted of the tumor and resection margins in most patients and additional intestinal biopsy specimens in some patients An extensive tissue library was available for patient This patient had surgical specimens taken over an 8-year period (1996 –2004) due to surveillance of long-standing CD and the development of metachronous neoplasia The clinical history of this patient is as follows The patient was male and had a 10-year history of CD and personal or family history of colorectal cancer In 2000, the patient developed a sigmoid cancer that was removed by a 7-cm segmental resection In 2001, a polypoid dysplastic lesion in the residual sigmoid colon was removed endoscopically In 2004, the patient developed perianal sepsis and became septic, and the first author became involved in the patient’s care A total proctocolectomy was performed The colon contained an invasive adenocarcinoma in the rectum, a dysplastic lesion in the transverse colon, and a right colon adenoma The patient was not on immunosuppressive medication Tissue Samples A complete list of tissue specimens from each patient that were subjected to molecular analysis is given in Supplementary Table For needle macrodissection, tissue was serially sectioned at a thickness of m: section was stained with H&E and underwent assessment by a pathologist, and sections 2– were mounted on plain glass slides For individual crypt analysis, tissue was serially sectioned at a thickness of m: sections 1–3 and 5–7 were mounted on PALM membrane slides (Zeiss) for laser capture, sections and were stained with H&E and underwent assessment by a pathologist, and sections –15 were mounted on plain glass slides for immunohistochemical analysis Section 16 was cut at a thickness of 40 m for image cytometry analysis Needle Macrodissection for Mutation Screening of Cancers Surgical resection specimens were used for mutation screening Plain glass slides were dewaxed and stained in methylene green (Vector Labs, Peterborough, United Kingdom) for approximately 10 seconds, rinsed in tap water, and dried at 37°C for hour Areas of cancer or dysplasia, or morphologically normal stromal tissue, were identified using the serial H&E slide and then carefully needle macrodissected from each of the plain glass slides Stromal tissue was subsequently used as constitutional DNA Dissected tissue was digested overnight at 65°C in 50 L of PicoPure proteinase K buffer (Arcturus FIELD CANCERIZATION IN CROHN’S DISEASE 10.e1 Bioscience, Mountain View, CA) Following digestion, proteinase K was inactivated by heating to 95°C for 10 minutes Samples were briefly centrifuged, and the DNA rich lysate was stored at Ϫ20°C until required Laser Capture Microdissection Laser capture PALM slides were dewaxed and then lightly stained with methylene green and air dried at 37°C for hour Individual crypts were selected for microdissection using the H&E slides as a guide The same crypt was then microdissected from each of the methylene green slides using a P.A.L.M laser capture microdissection system (Zeiss) Dissected crypts were digested overnight at 65°C in 12 L of PicoPure proteinase K buffer Tubes containing digestion buffer but no microdissection material were included as negative controls Following digestion, the proteinase K was deactivated and stored as described previously PCR and Sequencing All macrodissected samples were initially screened for somatic mutations in TP53 exons 5–9, KRAS exon (codons 12 and 13), and CDKN2A exon These genomic regions are known mutation hot spots in UC colorectal cancers Full primer details and reaction conditions are listed in Supplementary Table Identified mutations were then analyzed in the patient’s individual crypt lysates DNA was amplified using nested PCRs First-round PCRs were performed in an Omni PCR UV hood (Bioquell, Andover, England) to minimize the risk of contamination Only PCR products with an uncontaminated negative control tube were taken forward to sequencing Successfully amplified products were cleaned using EXOSAP-IT (USB-Web (part of Affymetrix) High Wycombe, UK) according to the manufacturer’s instructions and then sequenced using BigDye terminator cycle sequencing on an ABI 3100 sequencer (Applied Biosystems) Sequences were compared with the Cambridge reference sequence using 4Peaks software (MekenTosj.com, Amersterdam, The Netherlands) Mutations were confirmed as somatic using the Catalogue of Somatic Mutations in Cancer (www.sanger.ac.uk/cosmic), and putative polymorphisms were confirmed using Ensembl (www.ensembl.org) Full primer details and reaction conditions are listed in Supplementary Table LOH Analysis LOH analysis was performed on individual crypt/ gland lysates using a multiplexed microsatellite assay Nineteen highly polymorphic microsatellites, located on chromosomes 3p (FHIT), 5q (APC), 9p (p16INK4A), 17p (p53), 17q, and 18q (SMAD4), were amplified in separate reactions using a multiplex PCR kit (Qiagen) (See Supplementary Table 2) Microsatellites heterozygous in constitutional DNA were analyzed for LOH in individual 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 AQ: 18 615 616 617 618 619 AQ: 19 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 10.e2 636 637 638 639 AQ: 20 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 GALANDIUK ET AL crypts Amplification of constitutional and individual crypt lysate was performed to control for PCR variability Marker accession numbers and primer and reaction details are shown in Supplementary Table Primers were tagged at the 5= end with either a FAM or HEX fluorescent marker Successfully amplified PCR products were analyzed on an ABI 3100 sequencer (Applied Biosystems) and analyzed using Genotyper 2.5 software or Peakscanner v1.0 (Applied Biosystems) LOH was then considered present if the height of one allelic peak was more than twice that of the other, after normalizing the peak heights relative to the constitutional DNA For 5q and 17p markers, LOH was considered to be present only if shown by at least markers Microsatellite Instability Analysis Microsatellite instability was assessed using multiplexed assay for the BAT-25 and BAT-26 mononucleotide repeats.1 If a sample showed microsatellite instability, LOH analysis was not performed Image Cytometry The 40-m sections reserved for ploidy analysis were left waxed Areas of approximately mm2 of epithelium were needle dissected, using the H&E as a guide Image cytometry was performed using the Fairfield DNA Ploidy system (Fairfield Imaging) following Feulgen’s staining as previously described.2 Ploidy histograms were analyzed using the ABCDE approach described by Buhmeida et al.3 GASTROENTEROLOGY Vol xx, No x Immunohistochemistry Immunohistochemistry was performed for p53, -catenin, and lysozyme using a 3-step indirect avidinbiotin/peroxidase technique Sections were first dewaxed in xylene, taken to water through graded alcohols, and treated with 3% hydrogen peroxide to block endogenous peroxidases before microwaving in citrate buffer for antigen retrieval The primary layer was then applied: 1:50 p53 (Dako) for 60 minutes of incubation at room temperature, 1:100 -catenin (Transduction Labs) for 35 minutes, or 1:1000 lysozyme (Dako) for 35 minutes The secondary layer was 1:300 biotinylated rabbit anti-mouse (Dako) incubated for 35 minutes for p53 and -catenin staining or 1:500 biotinylated swine anti-rabbit (Dako) for lysozyme staining The tertiary layer was 1:500 streptavidin– horseradish peroxidase (Dako) incubated for 35 minutes Liquid DAB substrate (Dako) was used as the chromogen Sections were then counterstained in hematoxylin Supplementary References Stone JG, Tomlinson IP, Houlston RS Optimising methods for determining RER status in colorectal cancers Cancer Lett 2000; 149:15–20 Leedham SJ, Graham TA, Oukrif D, et al Clonality, founder mutations, and field cancerization in human ulcerative colitis-associated neoplasia Gastroenterology 2009;136:542–550.e6 Buhmeida A, Algars A, Ristamaki R, et al DNA image cytometry is a useful adjunct tool in the prediction of disease outcome in patients with stage II and stage III colorectal cancer Oncology 2006;70:427– 437 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 Month 2012 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 FIELD CANCERIZATION IN CROHN’S DISEASE Supplementary Table Primer Details and Reaction Conditions for Sequenced Loci Primer Sequence 5= to 3= p16-2A 1st F p16-2A 1st R p16-2B 1st F p16-2B 1st R p16-2A 2nd F p16-2A 2nd R p16-2B 2nd F p16-2B 2nd R KRAS 1st F KRAS 1st R KRAS 2nd F KRAS 2nd R p53-5 1st F p53-5 1st R p53-6 1st F p53-6 1st R p53-7 1st F p53-7 1st R p53-8 1st F p53-8 1st R p53-5 2nd F p53-5 2nd R p53-6 2nd F p53-6 2nd R p53-7 2nd F p53-7 2nd R p53-8 2nd F p53-8 2nd R GCTTCCTTTCCGTCATGC CAGGTACCGTGCGACATC CTGTTCTCTCTGGCAGGTCA TGTGCTGGAAAATGAATGCT CCTGGCTCTGACCATTCTGT CAGCTCCTCAGCCAGGTC CTTCCTGGACACGCTGGT TGGAAGCTCTCAGGGTACAAA GAGTTTGTATTAAAAGGTACTGGTGGA ATCAAAGAATGGTCCTGCAC TTTGATAGTGTATTAACCTTAT TATTAAAACAAGATTTACCTC CACTTGTGCCCTGACTTTCA GAGCAATCAGTGAGGAATCAGA AGAGACGACAGGGCTGGTT TGGAGGGCCACTGACAAC TGCTTGCCACAGGTCTCC GGTCAGAGGCAAGCAGAGG TTTTTAAATGGGACAGGTAGGA CACCCTTGGTCTCCTCCAC TCTGTCTCCTTCCTCTTCCTACA AACCAGCCCTGTCGTCTCT CAGGCCTCTGATTCCTCACT CTTAACCCCTCCTCCCAGAG CTTGGGCCTGTGTTATCTCC GTGTGCAGGGTGGCAAGT GCCTCTTGCTTCTCTTTTCC GCTTCTTGTCCTGCTTGCTT Reaction conditions 60/2/5 60/2/5 60/2/5 60/2/5 60/2/5 55/2/5 55/1/5 60/2/5 60/1/5 60/2/5 60/1/5 60/1/0 60/1/5 60/2/0 NOTE Naming nomenclature is as follows: gene-exon, PCR round, primer direction Large exons were subdivided into separate regions for amplification; a letter following the exon number denotes these regions (eg, p16-2A) For APC primers, the numbers not refer to exons but identify regions of the mutation cluster region Reaction condition nomenclature is annealing temperature/magnesium concentration (mmol/L)/addition of Qiagen Q solution 10.e3 Supplementary Table Microsatellite LOH Analysis Primers and Reaction Details Primer Multiplex D18S58 F D18S58 R D5S346 F D5S346 R D9S932 F D9S932 R D3S1300 F D3S1300 R Multiplex D17S250 F D17S250 R D18S474 F D18S474 R D17S1832 F D17S1832 R D3S1313 F D3S1313 R Multiplex D17S1176 F D17S1176 R D17S1678 F D17S1678 R D17S1881 F D17S1881 R Multiplex D9S942 F D9S942 R D5S2001 F D5S2001 R D17S1506E F D17S1506E R D5S489 F D5S489 R Multiplex D9S1752 F D9S1752 R D9S171 F D9S171 R D9S43 F D9S43 R D9S942 F D9S942 R D9S932 F D9S932 R BAT-25/26 BAT-26 F BAT-26 R BAT-25 F BAT-25 R Sequence 5= to 3= Conditions 57/0 GCTCCCGGCTGGTTTT GCAGGAAATCGCAGGAACTT ACTCACTCTAGTGATAAATCGGG AGCAGATAAGACAGTATTACTAGTT CTCCCTTTGTATTTCTGTTCTATT AAGCTATGATGGTGCCACC ACAAAGGAACGTCATGTGGTAGG GCTGTTTATTCTTCGTGGAATGCC 57/0 GGAAGAATCAAATAGACAAT GCTGGCCATATATATATTTAAACC CTCCACCCACTAGATGTCAG ACTTGCTTAAGCCTTGGACT ACGCCTTGACATAGTTGC TGTGTGACTGTTCAGCCTC TACTTTCCTTCAGATCCTTGG AACTAGGGGCCATGAATAAG 57/0 ACTTCATATACATATCACGTGC TCAATGGAGAATTACGATAGTG TTTGGGTCTTTGAACCCTTG CCACAACAAAACACCAGTGC CCCAGTTTAAGGAGTTTGGC TAGGGCAGTCAGCCTTGTG 57/5 GCAAGATTCCAAACAGTA CTCATCCTGCGGAAACCATT GCCAAGATGGTCTCGATCTC TCTGAACAGGTGATGGCAAC TGTGGGATGGGGTGAGATTTC CTGTTGGTCGGTGGGTTG GGGCTTTTGTGTTGTTTCTA GAAAACCCATAACCAGACTTG 57/0 AGACTACACAGGATGAGGTG GCAAGTCATAAGGGGATTTC AGCTAAGTGAACCTCATCTCTGTCT ACCCTAGCACTGATGGTATAGTCT TTCTGATATCAAAACCTGGC AAGGATATTGTCCTGAGGA GCAAGATTCCAAACAGTA CTCATCCTGCGGAAACCATT CTCCCTTTGTATTTCTGTTCTATT AAGCTATGATGGTGCCACC 57/0 TGACTACTTTTGACTTCAGCC AACCATTCAACATTTTTAACCC TCGCCTCCAAGAATGTAAGT TCTGCATTTTAACTATGGCTC NOTE Reaction condition nomenclature is annealing temperate/Q solution used in reaction Other conditions were performed according to the manufacturer’s instructions 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 10.e4 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 AQ: 21 799 800 801 802 803 GALANDIUK ET AL GASTROENTEROLOGY Vol xx, No x Supplementary Figure Genetic and protein analysis of the sigmoid cancer resected in patient in 2000 (A) H&E stain of cancer biopsy specimen showing moderately differentiated adenocarcinoma (original magnification 25ϫ) (B) p53 immunohistochemistry (original magnification 25ϫ) (C) -catenin immunohistochemistry (original magnification 25ϫ) (D) Methylene green–stained PALM membrane slide showing microdissected areas of the cancer (original magnification 25ϫ) (E) H&E stain showing moderately differentiated adenocarcinoma (original magnification 100ϫ) (F) p53 immunohistochemistry (original magnification 100ϫ) Strong nuclear expression of p53 is visible (G) -catenin immunohistochemistry (original magnification 100ϫ) The majority of epithelial cells show nuclear accumulation of -catenin (H) Sequencing showing the TP53 c.731GϾA mutation present throughout the cancer (I) LOH data for marker D5S346 indicating 5q LOH 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 Month 2012 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826 827 828 829 830 831 832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 FIELD CANCERIZATION IN CROHN’S DISEASE 10.e5 Supplementary Figure Genetic and protein analysis of the rectal cancer resected in patient in 2004 (A) H&E stain of cancer biopsy specimen showing moderately differentiated adenocarcinoma (original magnification 25ϫ) (B) H&E stain of dysplastic crypt (original magnification 100ϫ) (C) -catenin immunohistochemistry shows little nuclear accumulation of -catenin (original magnification 100ϫ) (D) p53 immunohistochemistry shows strong nuclear expression of p53 (original magnification 100ϫ) (E) Sequencing showing the TP53 c.742CϾT mutation that was present throughout the cancer (F) LOH data for marker D17S1881 indicating 17p LOH in cancer crypts 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826 827 828 829 830 831 832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 10.e6 860 861 862 863 864 865 866 867 868 869 870 871 872 873 874 875 876 877 878 879 880 881 882 883 884 885 886 887 888 889 890 891 892 893 894 895 896 897 898 899 900 901 902 903 904 905 906 907 908 909 910 911 912 913 914 915 GALANDIUK ET AL GASTROENTEROLOGY Vol xx, No x Supplementary Figure Image cytometry (A) Patient had transverse colon dysplasia resected in 2004 The dysplasia contained a c.775GϾT TP53 mutation and was diploid (B) Patient had transverse colon nondysplastic tissue resected in 2004 The tissue contained a c.742CϾT TP53 mutation and was aneuploid (C and D) Moderately and poorly differentiated regions, respectively, of the rectal cancer in patient in 2004 containing c.742CϾT TP53 mutation (C) The moderately differentiated region was diploid (D) The poorly differentiated region had developed aneuploidy Supplementary Figure Longitudinal analysis of clone spread over time in patient Tissue was collected for patient between 2004 and 2008 A nondysplastic small intestine biopsy specimen in 2004 showed a KRAS codon 12 mutation; a biopsy specimen years later was genetically wild type A KRAS codon 13 mutation was detected within intestinalized areas of the anal canal (fistula tracts) and was found with spatially distinct TP53 mutants, suggesting that the KRAS mutation was the founder mutation for the lesion Four years later, this same KRAS mutation was found throughout a cancer located within the perineal proctectomy scar, indicating that this pretumor clone had grown into a cancer Clonality between the cancer and the precursor lesion confirmed the presence of the same CDKN2A (p16) and TP53 mutations within both lesions 860 861 862 863 864 865 866 867 868 869 870 871 872 873 874 875 876 877 878 879 880 881 882 883 884 885 886 887 888 889 890 891 892 893 894 895 896 897 898 899 900 901 902 903 904 905 906 907 908 909 910 911 912 913 914 915 Month 2012 916 917 918 919 920 921 922 923 924 925 926 927 928 929 930 931 932 933 934 935 936 937 938 939 940 941 942 943 944 945 946 947 948 949 950 951 952 953 954 955 956 957 958 959 960 961 962 963 964 965 966 967 968 969 970 971 FIELD CANCERIZATION IN CROHN’S DISEASE 10.e7 Supplementary Figure Noninflamed mucosa did not contain mutant clones (A and B) Jejunum resection margin from patient showing no significant inflammation The crypts microdissected from this area were wild-type for the CDKN2A and KRAS mutations found in this patient’s jejunal cancer and its inflamed resection margins (A) H&E stain (original magnification 10ϫ) and (B) methylene green–stained laser capture slide (original magnification 10ϫ) showing microdissected crypt (C and D) Noninflamed terminal ileum tissue from patient Crypts microdissected from this area were all wild type for the TP53 mutation found in the patient’s cecal cancer (C) H&E stain (original magnification 10ϫ) and (D) methylene green–stained laser capture slide (original magnification 10ϫ) 916 917 918 919 920 921 922 923 924 925 926 927 928 929 930 931 932 933 934 935 936 937 938 939 940 941 942 943 944 945 946 947 948 949 950 951 952 953 954 955 956 957 958 959 960 961 962 963 964 965 966 967 968 969 970 971 ... mutation in functional small intestinal and colon crypts (A) H&E section showing inflamed terminal ileum mucosa with crypt/villous distortion and an increase in inflammatory cells and intraepithelial... Patients and had cancer in the small intestine, whereas patients 1, 5, and had colon cancer, indicating that pretumor clone growth occurred in both intestinal compartments The remaining patients (patients... in the CD bowel mirrors that reported in the International Agency for Research on Cancer database for the intestine at large However, given that other gastrointestinal cancers arising in an inflammatory