1. Volume 129, Issue 3, Pages 1047-1059 (September 2005)
Defective DNA Mismatch Repair Determines a Characteristic Transcriptional Profile in Proximal Colon Cancers 
Massimiliano di Pietro, Jacob Sabates Bellver, Mirco Menigatti, Fridolin Bannwart, Annelies Schnider, Anna Russell, Kaspar Truninger, Josef Jiricny, Giancarlo Marra 
Gastroenterology 
Volume 129, Issue 3, Pages (September 2005)
DOI: /j.gastro
Copyright © 2005 American Gastroenterological Association Terms and Conditions

2. Figure 1
Unsupervised hierarchical clustering of normal colon mucosa and colon cancer samples based on analysis with the Affymetrix gene set after exclusion of unexpressed genes (see supplemental material online at The 5858 probes plotted on the y-axis are color coded to indicate their expression levels relative to the median level for the gene across the entire sample set (blue, lower; red, higher). The 35 tissue samples represented on the x-axis include 10 normal mucosa samples (green bars), 12 MMR-deficient cancers (red bars), and 13 MMR-proficient cancers (blue bars).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

3. Figure 2
Principal component analysis of the 25 proximal colon cancers and the 14 colon cancer cell lines. (See supplemental material online at for details on principal component analysis methodology.) (A) Normalized component-1 output values (y-axis, log-scale) for the 25 cancer specimens (x-axis, numbered as in Table 1). The difference between values for MMR-proficient and MMR-deficient cancers was highly significant (t = 4.70; P < .0001). (B) Unsupervised hierarchical clustering of the 25 cancers based on the component-1 gene set, that is, the 151 genes (160 probes) with significant principal component analysis values (greater than 0.7 and less than −0.7) in component 1. With 2 exceptions, MMR-proficient cancers 3 and 8 (red bars), the cancers formed 2 clusters that coincided with MMR-status subgroups. (C) Normalized component-2 output values (y-axis, log-scale) for the 25 cancers (x-axis, numbered as in Table 1). There was a highly significant difference between values for the lymph node–positive and lymph node–negative cancers (t = 5.27; P < .0001). (D) Component-1 and (E) component-2 output values (y-axes, log-scale) for the 14 colon cancer cell lines (x-axes). Components 1 and 2 accounted for 20% and 13%, respectively, of the total variance in the cell line data set. In both cases, MMR-deficient and MMR-proficient cell lines were distinguished by significantly different output values (component 1: t = 2.26, P = .043; component 2: t = 3.88, P = .0022).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

4. Figure 3
Supervised hierarchical clustering of colon cancers and colon cancer cell lines classified on the basis of their MMR status. (A) Clustering of 25 colon cancers by the MMR-status signature gene set (100 genes, 106 probes) shown in Table 3. (B) Clustering of 14 colon cancer cell lines by the 66 genes shown in Supplementary Table 2, which can be considered a cell line–specific version of the MMR-status signature set. (C) Clustering of colon cancers and cell lines by the MMR-related epithelial cell signature gene set shown in Table 4. Sample tree: MMR-proficient cell lines in light blue, MMR-proficient cancers in dark blue, MMR-deficient cell lines in orange, and MMR-deficient cancers in red.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

5. Figure 4
Selection of the MMR-related tumorigenesis signature gene set from the MMR-status signature gene set. (A) The light gray circle represents the 682 genes whose expression levels in MMR-deficient cancers were significantly increased or decreased, relative to those in normal mucosa (NM), with fold changes (FC) of ≥2.0. (The 10 normal mucosa samples were considered as a homogeneous group because their variability was much less marked than that observed among the cancer specimens, as shown in Figure 1). The white circle represents 759 genes displaying significantly altered expression (no FC restrictions) in MMR-proficient cancers with respect to normal mucosa. (Elimination of the FC cutoff in this selection allowed us to identify MMR-deficient–specific changes with greater accuracy.) The intersection of the 2 circles represents the 383 genes displaying similar alterations in MMR-deficient and MMR-proficient cancers with respect to normal mucosa. Exclusion of these genes leaves 299 genes whose expression was altered (relative to normal mucosa) exclusively in MMR-deficient cancers. (B) A similar procedure was used to identify 190 genes with significant expression changes (relative to normal mucosa) that were confined exclusively to MMR-proficient cancers. (C) The light gray circles here represent the sets of 299 and 190 genes obtained in A and B, while the dark gray circle represents the 100 genes making up the MMR-status signature (Table 3). Gene set characteristics in terms of differences between transcriptional levels in MMR-deficient cancers, MMR-proficient cancers, and normal mucosa are summarized in the small rectangles; differences are classified as significant (S) or nonsignificant (NS). Overlap between the light gray circle on the left and the dark gray circle represents 21 genes whose expression levels in MMR-deficient cancers were significantly different (FC ≥2.0) from those observed in normal mucosa and also significantly different from those of MMR-proficient cancers. Intersection of the dark gray circle and the light gray circle on the right defines 18 other genes with expression levels in MMR-proficient cancers that differed significantly (FC ≥2.0) from those of normal mucosa and also from those of MMR-deficient cancers. These 39 genes represent a subset of the MMR-status signature gene set, which we refer to as the MMR-related tumorigenesis signature.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

6. Figure 5
4-1BBL messenger RNA (mRNA) and protein expression in MMR-deficient and MMR-proficient colon cancer cell lines. (No data are shown for 5 of the cell lines, which could not be reliably analyzed with flow cytometry.) (A) Gray bars represent mRNA levels based on raw signals (y-axis) detected in MMR-deficient (x-axis; left) and MMR-proficient (x-axis; right) cell lines with the Affymetrix U95v2 microarray. (B) Gray bars represent mRNA levels for the 4-1BBL coding region in MMR-deficient (left) and MMR-proficient (right) cell lines determined by quantitative reverse-transcription polymerase chain reaction (RT-PCR); white bars represent corresponding levels for amplification of the control housekeeping gene GAPDH. The 4-1BBL coding region was amplified with primers complementary to the exon 2/exon 3 boundary (forward) and to the central region of exon 3 (reverse). The number of cycles (y-axis) at the beginning of the log-phase amplification is inversely correlated with the abundance of the transcript. (C) Gray bars represent cytoplasmic membrane expression of 4-1BBL polypeptide assessed by flow cytometry. Jurkat and Raji cells (white bars) were used as negative and positive controls, respectively, and expression values in colon cancer cell lines were normalized to that of Raji cells. All RT-PCR and flow cytometry assays were performed in duplicate.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions