BackgroundCancers 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.PurposeTo determine whether reduced expression of DNA repair proteins Pms2, Ercc1 and Xpf (pairing partner of Ercc1) are early steps in progression to colon cancer.ResultsTissue 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.ConclusionsThe 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.

AIM:To investigate whether deficiency of expression of cytochrome c oxidase I (CcOI) in colonic crypts is associated with colon cancer. METHODS:The pattern and level of expression of CcOI in non-neoplastic colonic crypts, and in dysplastic tissues, was assessed using standard immunohistochemical methods. Biopsies were obtained from individuals undergoing colonoscopies for screening purposes or for a medically indicated reason. Tissue samples were also obtained from surgical colonic resections. Samples from resections were taken from colonic mucosa 1 and 10 cm from tumors and from the tumors themselves. Samples were evaluated for frequency of crypts with reduced or absent expression of CcOI. In most crypts the loss was apparent throughout the entire crypt, while in a small minority the loss was segmental. The strong immunoreactivity using this monoclonal antibody makes the scoring unambiguous. The percent of crypts with reduced or absent expression of CcOI or (infrequent) segmented loss of expression was then calculated. Data analyses were performed using SPSS statistical package 17.0. RESULTS:The average frequency of CcOI deficient crypts (CcOI-DC) is low in individuals between 20 and 39 years of age, with 0.48% ± 0.40% CcOI-DC for women and 1.80% ± 0.35% for men. CcOI-DC increases after age 40 years, so that between the ages of 40 and 44 years the average frequency of CcOI-DC goes up to 5.89% ± 0.84% in women and 2.15% ± 1.27% in men. By 80-84 years of age, the average frequency of CcOI-DC goes up in women to 15.77% ± 0.97% and in men to 22.6% ± 0.65%. The increases in CcOI-DC from ages 40-44 years compared to 80-84 years in women and men are significantly different with P < 0.01. For women over age 60 years, deficiency of CcOI expression is greater in those women who have had a cancer in their colon. The frequency of CcOI-DC, measured in men, increased in tissues adjacent to colon cancer, being 4.03% ± 0.27% in individuals free of neoplasia in the age range 55-64 years

In carcinogenesis, the "field defect" is recognized clinically because of the high propensity of survivors of certain cancers to develop other malignancies of the same tissue type, often in a nearby location. Such field defects have been indicated in colon cancer. The molecular abnormalities that are responsible for a field defect in the colon should be detectable at high frequency in the histologically normal tissue surrounding a colonic adenocarcinoma or surrounding an adenoma with advanced neoplasia (well on the way to a colon cancer), but at low frequency in the colonic mucosa from patients without colonic neoplasia.Using immunohistochemistry, entire crypts within 10 cm on each side of colonic adenocarcinomas or advanced colonic neoplasias were found to be frequently reduced or absent in expression for two DNA repair proteins, Pms2 and/or ERCC1. Pms2 is a dual role protein, active in DNA mismatch repair as well as needed in apoptosis of cells with excess DNA damage. ERCC1 is active in DNA nucleotide excision repair. The reduced or absent expression of both ERCC1 and Pms2 would create cells with both increased ability to survive (apoptosis resistance) and increased level of mutability. The reduced or absent expression of both ERCC1 and Pms2 is likely an early step in progression to colon cancer.DNA repair gene Ku86 (active in DNA non-homologous end joining) and Cytochrome c Oxidase Subunit I (involved in apoptosis) had each been reported to be decreased in expression in mucosal areas close to colon cancers. However, immunohistochemical evaluation of their levels of expression showed only low to modest frequencies of crypts to be deficient in their expression in a field defect surrounding colon cancer or surrounding advanced colonic neoplasia.We show, here, our method of evaluation of crypts for expression of ERCC1, Pms2, Ku86 and CcOI. We show that frequency of entire crypts deficient for Pms2 and ERCC1 is often as great as 70% to 95% in 20 cm long areas surrounding a colonic neoplasia, while frequency of crypts deficient in Ku86 has a median value of 2% and frequency of crypts deficient in CcOI has a median value of 16% in these areas. The entire colon is 150 cm long (about 5 feet) and has about 10 million crypts in its mucosal layer. The defect in Pms2 and ERCC1 surrounding a colon cancer thus may include 1 million crypts. It is from a defective crypt that colon cancer arises. Video LinkThe video component of this article can be found at http://www.jove.com/video/1931/ Protocol Preparing tissues to be viewed on slides 1. A colonoscope can be passed into the colon through the rectum. A colonoscope is a long tube that has illumination and a video camera at the head, the ability to pass air into the colon to blow it up like a long balloon to allow better visualization of the colon, and has a passage for biopsy forceps that can extend beyond the video camera to pinch off a 3-5 millimeter area of the inner surface "skin" or mucosal layer of the colon to give us a biopsy specimen. The biopsy forceps can a...

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