Volume 136, Issue 2, Pages e6 (February 2009)

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Volume 136, Issue 2, Pages 542-550.e6 (February 2009) Clonality, Founder Mutations, and Field Cancerization in Human Ulcerative Colitis– Associated Neoplasia  Simon J. Leedham, Trevor A. Graham, Dahmane Oukrif, Stuart A.C. McDonald, Manuel Rodriguez–Justo, Rebecca F. Harrison, Neil A. Shepherd, Marco R. Novelli, Janusz A.Z. Jankowski, Nicholas A. Wright  Gastroenterology  Volume 136, Issue 2, Pages 542-550.e6 (February 2009) DOI: 10.1053/j.gastro.2008.10.086 Copyright © 2009 AGA Institute Terms and Conditions

Figure 1 Laser capture dissection. (A, i) Suitable crypts for dissection were identified using H&E slides. (A, ii and iii) Individual crypts were microdissected from serial methylene green–stained sections mounted on laser capture slides. (A, iv) Dissected crypt on the cap of the laser capture tube. (B, i and ii) Individual crypts (black arrows) were dissected across a specimen and numbered from left to right across the lesion. Gastroenterology 2009 136, 542-550.e6DOI: (10.1053/j.gastro.2008.10.086) Copyright © 2009 AGA Institute Terms and Conditions

Figure 2 Topographic clonal map from lesion 2 with phenotypic heterogeneity. (A) Colectomy specimen H&E and (B) laser capture slide. (C, i–v) The tissue was subdivided into phenotypically graded populations. (D) Topographic clonal map of crypts dissected from left to right across the lesion. Each column represents the genetic analysis and mutation burden of a single crypt. Different p53 point mutations are denoted by different shades of blue-colored boxes. The individual mutation is detailed in the column on the left. In this case the 2 p53 point mutations were from a single allele (supplementary Figure 1; see supplementary material online at www.gastrojournal.org), with the remaining allele undergoing allelic loss represented by orange boxes. White boxes are wild-type. Membranous β-catenin staining is represented by an m (supplementary Figure 1A; see supplementary material online at www.gastrojournal.org), and lack of nuclear p53 immunostaining by a minus sign. Considerable phenotypic heterogeneity was seen in this sample with small areas of low-grade dysplasia arising from surrounding nondysplastic chronically inflamed and hyperplastic crypts. However, all crypts dissected from across the block were clonal for p53 mutations. Image cytometry ploidy analysis showed an aneuploid population (histogram grade C) throughout the specimen (supplementary Figure 3A, iii; see supplementary material online at www.gastrojournal.org). Gastroenterology 2009 136, 542-550.e6DOI: (10.1053/j.gastro.2008.10.086) Copyright © 2009 AGA Institute Terms and Conditions

Fig. 3 Topographic clonal map from lesion 3 with p53 immunostaining. (A) H&E and laser capture slide of the colectomy specimen with high-grade and low-grade dysplastic areas. (B, i) High-grade dysplasia; (B, ii) magnification, 100×; and (B, iii) magnification, 400× of p53 immunostaining showing strong nuclear positivity. (C, i) H&E of transition zone between high-grade and low-grade dysplasia. (C, ii) magnification, 100×; and (C, iii) magnification, 250×. Sharp cut-off of nuclear p53 immunoreactivity concurring with the genetic analysis of multiple crypts from this region. (D, i) Low-grade dysplasia; (D, ii) magnification, 100×; and (D, iii) magnification, 400× of negative nuclear p53 immunostaining in low-grade area. (E) Topographic clonal map for the colectomy specimen pictured. Each column represents the genetic analysis of a single crypt. Different p53 point mutations are denoted by different shades of blue-colored boxes. In this case bi-allelic point mutation were identified (supplementary Figure 2; see supplementary material online at www.gastrojournal.org). K-RAS mutation is represented by a red box. Individual mutations are detailed in the column on the left. White boxes are wild-type. The same K-RAS mutation was found in all crypts from across the block, whereas p53 point mutations were seen only in the high-grade dysplastic half of the lesion. This strongly supports a founder K-RAS mutation with subsequent p53 inactivation occurring in a genetically and phenotypically advanced subclone. Nuclear p53 immunostaining is denoted by plus or minus signs. Membranous β-catenin is denoted by m and is depicted in supplementary Figure 3B (see supplementary material online at www.gastrojournal.org). Gastroenterology 2009 136, 542-550.e6DOI: (10.1053/j.gastro.2008.10.086) Copyright © 2009 AGA Institute Terms and Conditions

Supplementary Figure 1 Monoallelic p53 mutations in lesion 2. Cloning allows the amplification of a single DNA fragment and thus the identification of a single allelic product. (A) The wild-type p53 sequence. (B) The originally obtained sequence identifying 2 heterozygous point mutations. Red, c.586C>T, p.R196X nonsense mutation; blue, c.614A>G, p.Y205C missense mutation. (C) Individual allele sequence after cloning showing 1 completely wild-type allele and 1 mutant allele with both mutations present. This shows that both mutations were from a single allele and suggests that the second mutation (c.614A>G, p.Y205C) is a closely linked hitchhiker on the back of the more selectively advantageous nonsense mutation (c.586C>T, p.R196X). Gastroenterology 2009 136, 542-550.e6DOI: (10.1053/j.gastro.2008.10.086) Copyright © 2009 AGA Institute Terms and Conditions

Supplementary Figure 2 Bi-allelic p53 mutations in lesion 3. (A) The wild-type p53 sequence. The areas of interest are separated by 16 codons that are represented by a dashed line. (B) The originally obtained sequence identifying 2 heterozygous point mutations. Red, c.725G>T, p.C242F missense mutation; blue, c.772G>C, p.E258Q missense mutation. (C) Individual allele sequences after cloning showing 1 mutation present on each allele, showing a true bi-allelic point mutation. Gastroenterology 2009 136, 542-550.e6DOI: (10.1053/j.gastro.2008.10.086) Copyright © 2009 AGA Institute Terms and Conditions

Supplementary Figure 3 β-catenin staining and image cytometry histograms. (A, i) Low-power and (A, ii) high-power view of membranous β-catenin staining of dysplastic crypts from lesion 2. (A, iii) Image cytometry histogram (grade C) showing 2C peak on the left marked by internal control lymphocyte nuclei colored blue. A clear noneuploid peak is visible between the 2C and 4C gates. (B, i) Low-power and (B, ii) high-power view of membranous β-catenin staining of dysplastic crypts from lesion 3. (B, iii) Image cytometry histogram (grade A) showing diploid 2C peak marked by internal control lymphocyte nuclei colored blue. Gastroenterology 2009 136, 542-550.e6DOI: (10.1053/j.gastro.2008.10.086) Copyright © 2009 AGA Institute Terms and Conditions