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Deficient Expression of DNA Repair Enzymes in Early Progression to Sporadic Colon Cancer

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Deficient Expression of DNA Repair Enzymes in Early Progression to Sporadic Colon Cancer

Alexander Facista et al. Genome Integr.

Abstract

Background: Cancers often arise within an area of cells (e.g. an epithelial patch) that is predisposed to the development of cancer, i.e. a "field of cancerization" or "field defect." Sporadic colon cancer is characterized by an elevated mutation rate and genomic instability. If a field defect were deficient in DNA repair, DNA damages would tend to escape repair and give rise to carcinogenic mutations.

Purpose: To determine whether reduced expression of DNA repair proteins Pms2, Ercc1 and Xpf (pairing partner of Ercc1) are early steps in progression to colon cancer.

Results: Tissue biopsies were taken during colonoscopies of 77 patients at 4 different risk levels for colon cancer, including 19 patients who had never had colonic neoplasia (who served as controls). In addition, 158 tissue samples were taken from tissues near or within colon cancers removed by resection and 16 tissue samples were taken near tubulovillous adenomas (TVAs) removed by resection. 568 triplicate tissue sections (a total of 1,704 tissue sections) from these tissue samples were evaluated by immunohistochemistry for 4 DNA repair proteins. Substantially reduced protein expression of Pms2, Ercc1 and Xpf occurred in field defects of up to 10 cm longitudinally distant from colon cancers or TVAs and within colon cancers. Expression of another DNA repair protein, Ku86, was infrequently reduced in these areas. When Pms2, Ercc1 or Xpf were reduced in protein expression, then either one or both of the other two proteins most often had reduced protein expression as well. The mean inner colon circumferences, from 32 resections, of the ascending, transverse and descending/sigmoid areas were measured as 6.6 cm, 5.8 cm and 6.3 cm, respectively. When combined with other measurements in the literature, this indicates the approximate mean number of colonic crypts in humans is 10 million.

Conclusions: The substantial deficiencies in protein expression of DNA repair proteins Pms2, Ercc1 and Xpf in about 1 million crypts near cancers and TVAs suggests that the tumors arose in field defects that were deficient in DNA repair and that deficiencies in Pms2, Ercc1 and Xpf are early steps, often occurring together, in progression to colon cancer.

Figures

Figure 1
Figure 1
Schematic diagram of colonic mucosa indicating progression of a field defect to colon cancer. The gray area within the right-hand set of irregular concentric areas indicates a colon cancer. The outermost irregular concentric areas indicate initial defects with a selective advantage. A next smaller concentric area indicates a secondary mutation or epimutation giving a further selective advantage, while still smaller areas indicated further mutations or epimutations with still further selective advantages.
Figure 2
Figure 2
Sequential sections of the same crypt with high expression of Pms2 (A), Ercc1 (B) and Xpf (C). This crypt, from the biopsy of a 58 year old male patient who never had colonic neoplasia, shows high expression (brown) in absorptive cell nuclei throughout most of the crypt for each of the proteins. Note that Pms2 and Xpf expression (in panels A and C) are each reduced or absent in the nuclei of cells at the top of the crypt and within the surface of the colonic lumen between crypts. Images taken at 200×.
Figure 3
Figure 3
Single crypts having deficient expression for Pms2 (A), Ercc1 (B) or Xpf (C). These crypts are from a histologically normal area of a colon resection of a male patient who had an adenocarcinoma in the sigmoid colon. When Pms2 is deficient, typically all the cells of the crypt have low or absent expression for Pms2 (A). When Ercc1 is deficient, cells of the "body" have reduced or absent expression of Ercc1 but cells of the "neck" and "surface" usually have high expression for Ercc1 (B). When Xpf is deficient, cells of the body have reduced or absent expression of Xpf, but cells of the neck region often have high expression of XPF. Cells at the surface may have reduced expression (C) or may have high expression for Xpf (not shown here but see Figure 4). Images taken at 200×.
Figure 4
Figure 4
Sequential tissue sections with all crypts having reduced "body" expression of Pms2 (A), Ercc1 (B) and Xpf (C). These crypts are from a histologically normal area of a colon resection of a male patient who had an adenocarcinoma in the sigmoid colon (same tissue as in Figure 3). There is high expression of Ercc1 at the neck and surface of the crypts (B). There is also some expression of Xpf at the neck of each crypt but not in the clonic lumen in this area of tissue (C). For Pms2 (A), there is reduced expression in the body, the neck and surface for all epithelial cells. Images taken at 200×.
Figure 5
Figure 5
Tissue with fissioning crypts stained for Pms2 (A), Ercc1 (B) and Xpf (C). These sequential sections were from histologically normal tissue marginal to a resected sigmoid adenocarcinoma of a female patient. There appear to be fissioning crypts in this area of tissue, indicated by arrows. These fissioning crypts have reduced expression for each of the three proteins, Pms2, Ercc1 and Xpf. Most of the other crypts in this area have high expression of Pms2, Ercc1 and Xpf in their cell nuclei. These fissioning crypts may constitute a small patch of DNA repair-defective crypts that are increasing in patch size by crypt fission. Images taken at 200×.
Figure 6
Figure 6
Sequential tissue sections with discordant expression of Pms2, Ercc1 and Xpf. These crypts are from the biopsy of a 71 year old female patient who had a 1 cm adenocarcinoma. While Pms2 and Xpf have reduced expression in this tissue area, Ercc1 has high expression. In this mucosal area, one crypt is apparently fissioning into 3 crypts. Images taken at 200×.
Figure 7
Figure 7
Absorptive cells and goblet cells in crypts express DNA repair proteins differently. These three panels show parts of single crypts from a biopsy of a male patient who had a 1.5 cm tubular adenoma, stained by IHC for Pms2 (A), Ercc1 (B) and Xpf (C). Goblet cells of the crypts have a large "balloon like" region containing mucin granules (cytoplasmic white areas under these staining conditions). The other cells in the crypts are absorptive cells. Arrows indicate some nuclei of goblet cells (on the outer edges of the crypts) that have reduced or absent expression for Pms2, Ercc1 and Xpf. Images taken at 1000×.
Figure 8
Figure 8
Two pairs of entire tissue sections with consistent protein expression throughout all the crypts. Panels A and C are sequential tissue sections from a biopsy taken from a 57 year old male patient with current tubulovillous adenomas and a history of colon cancer. Panels B and D are non-sequential tissue sections from a tissue sample taken from a resection from a male patient who had a carcinoma in the sigmoid. Panels A and B were stained for Pms2, panels C and D were stained for XPF. All crypts in each tissue section had similar levels of protein expression, with high (A, C) or low (B, D) protein expression. Images B and D were tiled, since the entire tissue section could not be captured in one field of view. Images taken at 40×.
Figure 9
Figure 9
A tissue section immunostained for Ku86. A tissue section from a biopsy of a 53 year old male patient who never had a colonic neoplasia. A small patch of three crypts with reduced expression for Ku86 is shown by arrows.
Figure 10
Figure 10
Tissue samples evaluated with respect to expression of Pms2, Ercc1, Xpf or Ku86. Entire tissue sections were evaluated with respect to all crypt absorptive cells seen in the tissue or within all cells of epithelial origin within a cancer. Pms2, Ercc1 and Xpf were evaluated in triplicate sequential 4 micron tissues sections of the same tissue samples. Ku86 tissue samples were from similar, but not identical, tissues to those evaluated for Pms2, Ercc1 and Xpf. Tissue samples from patients at different risks for colon cancer, or from colon segments resected because of colon cancer, were labeled A, B, C, D, E, F, G and the number of tissue samples, from different patients, evaluated for protein expression are shown in parentheses under their label. The tissue samples were from colonic biopsies from patients who had (A) never had an adenoma; (B) 1 to 3 adenomas < 1 cm; (C) advanced neoplasia with either adenoma > 1 cm, villous adenoma, or adenoma with dysplasia; (D) previous colon cancer; or from colonic resections with (E) tissue sample 1 to 10 cm distant from an adenocarcinoma; (F) area marginal to the tumor; or (G) epithelial origin cells within the adenocarcinoma.
Figure 11
Figure 11
Each dot represents the joint evaluation of a tissue sample for 2 DNA repair proteins. The colors of the dots a, b, c, d, e, f, g represent tissues of groups A, B, C, D, E, F, G respectively, defined in Figure 10. Black lines represent mean values of expression of tissue sections from group A while red lines represent values of expression which are -2σ less than the mean values. Numbers in the small boxes show the mean and -2σ expression values. Dots that are below and to the left of the red lines have significantly reduced expression for both proteins being evaluated.
Figure 12
Figure 12
A Venn diagram illustrating the level of concordance of deficiencies of Pms2, Ercc1 and XPF. Entire tissue sections were evaluated with respect to all crypt absorptive cells seen in the tissue or within all cells of epithelial origin within a cancer. Pms2, Ercc1 and Xpf were evaluated in triplicate sequential 4 micron tissues sections of the same tissue samples.
Figure 13
Figure 13
Part of a sigmoid area colon resection, opened longitudinally, and showing a colon cancer. Tissue samples were obtained from such freshly resected colon segments. A ruler was placed across the opened colon segment to determine the inner circumference of the opened colon segment.
Figure 14
Figure 14
Percent of crypts with high expression of Pms2, Ercc1 or Xpf. Tissue samples were taken at the indicated distances from a colon cancer. The distances are -10, -3 or -1 cm on the proximal side and 1, 3 and 10 cm on the distal side of a resection from patient C1 (from Table 1). The symbols indicate the percent of crypts showing high expression of Pms2, Ercc1 or Ku86 in each tissue sample.
Figure 15
Figure 15
Diagram of the human lower gastrointestinal tract. This diagram illustrates the locations of the regions referred to in Table 2. Distances from the anal verge are shown in centimeters.

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