Crypt restricted heterogeneity of goblet cell mucus glycoprotein in histologically normal human colonic mucosa: a potential marker of somatic mutation

Abstract

Current evidence from experimental animals suggests that the colonic crypt is a clonal unit derived from, and maintained by, a single cell (Griffiths et al., 1988). Treatment of normal female TO strain mice with the colon-specific carcinogen dimethylhydrazine (DMH) results in a histochemically demonstrable loss of activity of the X-linked enzyme glucose-6-phosphate dehydrogenase (G6PD) in single, randomly distributed colonic crypts. This phenotypic change is uniform within the affected crypts, and its frequency is related to the dose of DMH treatment, strongly suggesting that it is the result of a carcinogen induced mutation at the G6PD locus on the active X-chromosome of a single primary crypt stem cell (Griffiths et al., 1988).A similar crypt-restricted phenotypic change has been demonstrated in normal human large bowel mucosa using the mild periodic acid-Schiff (mPAS) technique (Sugihara & Jass, 1986). This histochemical method distinguishes between 0-acetylated sialomucins, which are mPAS-negative, and non-0-acetylated sialomucins which stain a magenta colour (Veh et al., 1982). Sugihara and Jass (1986) found that in most human colons the goblet cell mucus is O-acetylated and mPAS-negative but that in a small proportion of individuals it is non-O-acetylated and diffusely mPAS-positive. However, in some cases with mPAS-negative colonic mucus glycoproteins they observed scattered individual mPAS-positive crypts. They were uncertain of the significance of this but interpreted it as a 'functional metaplasia'. A similar phenomenon has been identified by Hughes et al. (1986), who described a focal loss of immunoreactivity to a monoclonal antibody (3NM) directed against colonic goblet cells, again confined to scattered crypts in the human large intestine.Because the crypt-restricted phenotypic alterations described in these two reports are similar to the mutageninduced loss of G6PD activity in the colonic crypts of experimental mice, we considered that they may result from crypt stem cell mutation. If this were correct, the frequency of affected crypts would increase with exposure to environmental mutagens, and be age-related. The change might also occur more frequently in subjects with colorectal cancer. We have investigated this using the mPAS technique on histologically normal areas of resection specimens of the sigmoid colon and upper rectum from 30 adults (mean age 71.8 years, range 55-91) with primary large intestinal adenocarcinoma, 30 age-matched controls (mean age 71.3 years, range 52-90) with benign conditions of the sigmoid colon (diverticular disease 27, volvulus 3), and 18 infants or children (mean age 25 months, range 2 days to 9 years) with Hirschsprung's disease (16), intestinal atresia (1) or colonic duplication (1). One block of formalin-fixed, paraffin-embedded tissue from each resection margin of the colectomy specimens was taken and 5 im histological sections were cut at 50pm (Veh et al., 1982). In nine cases (three from each patient group) two adjacent levels were cut, one being stained by t...

Significant increase of colonic mutated crypts correlates with age in sporadic cancer and diverticulosis cases, with higher frequency in the left‐ than right‐side colorectum

“…2), suggesting that mutated crypts appear after birth and increase with age. This is in line with an earlier report by Fuller et al (7) in which the colonic mucosa of 18 infants and children revealed only two patterns of staining reaction, either uniformly negative or uniformly positive mPAS staining, indicating no demonstration of mutated crypts. This tendency would explain the increase in colorectal cancer development with age, (16) supporting multistep carcinogenesis.…”

“…This tendency would explain the increase in colorectal cancer development with age, (16) supporting multistep carcinogenesis. (4) According to the previous report, (7) no difference was found in the mutation frequencies of colonic crypts between age-matched individuals with sporadic colorectal cancer and benign colonic conditions. Therefore, it is worthwhile to see the age influence on mutation frequencies in the present study, although non-cancerous mucosa is not completely normal in cases with sporadic colorectal cancer.…”

“…The numbers of crypt profiles adjacent to the muscularis mucosa present in one central step section in each case were counted manually by a single observer (IO), according to the methods of Fuller et al (7) and Campbell et al (8) The total number of crypt profiles present was then calculated by multiplying the number of step sections by the count for the central section. For mPAS staining, 4-µm sections were cut from paraffin-embedded blocks and step sections were taken at 80-µm intervals to obtain the necessary size of sample while reducing the chance of counting the same crypt in adjacent sections.…”

Crypt-Restricted Loss and Decreased Protein Expression of Cytochrome c Oxidase Subunit I as Potential Hypothesis-Driven Biomarkers of Colon Cancer Risk

“…Because of the crypt-restricted loss of ability to acylate O-sialomucin, the goblet cells in an entire single crypt stain positively with mild periodic acid Schiff (38), or negatively by the periodate-borohydride/potassium hydroxide saponification/periodic acid Schiff method which stains O-acetyl sialic acid residues (39). This crypt-restricted staining with mild periodic acid Schiff was observed after X-irradiation therapy for rectal cancer (31) (38).…”

Comparative lesion sequencing provides insights into tumor evolution

“…This approach yields T pot , defined as the time between cell divisions in the absence of cell death. Several hundred colorectal cancers have been evaluated by this method, with T pot measured as Ϸ4 days (16)(17)(18)(19)(20)(21). By using this figure for the cell division rate and the mutational data reported in ref.…”