Abstract
Background
Previously, we indicated that stromal genetic instability might contribute to tumorigenesis of both sporadic and ulcerative colitis associated colorectal adenocarcinomas. Considering the established adenoma‐adenocarcinoma sequence, in this study we analysed genetic instability in colorectal adenoma cells and surrounding stroma.
Methods
In 164 colorectal tumours (34 hyperplastic polyps, 38 tubular adenomas with low grade dysplasia (TA‐L), 51 tubular adenomas with high grade dysplasia (TA‐H), and 41 invasive carcinomas), epithelial and stromal genetic instability with National Cancer Institute standard microsatellite markers and chromosome 17 (Chr17) markers, were analysed by a combination of laser capture microdissection and GeneScan approaches.
Results
While frequencies of both loss of heterozygosity (LOH) and microsatellite instability (MSI) were extremely low in hyperplastic polyps, LOH in tubular adenomas was detected in both epithelial (TA‐L 13.2%, TA‐H 27.5%) and stromal (5.3% and 5.9%, respectively) elements, along with MSI (5.3% and 13.7%, and 5.3 and 5.9%, respectively). Frequencies of epithelial alterations were higher in TA‐H than in TA‐L, and greatest in the carcinoma group. On the other hand, frequencies of stromal LOH or MSI were almost constant (5.3% ∼ 17.1%, 5.3% ∼ 17.1%, respectively) in adenomas and invasive carcinomas. In addition, p53 was found to be significantly overexpressed in a greater proportion of TA‐L with LOH than in those without genetic instability.
Conclusion
The results indicate the presence of genetic alterations in stroma from an early stage of carcinogenesis, accompanied by stepwise increasing genetic instability of epithelia with progression to cancer. Thus microenvironmental changes due to genetic alteration in Chr17 markers in stromal cells may play an important role in colon adenoma and adenocarcinoma development.
Keywords: genetic instability, stroma, colorectum, adenoma, tumorigenesis
Interactions between epithelial and mesenchymal cells in various organs play important roles in development,1,2 differentiation,3,4 and growth.3,4 However, the majority of studies on colorectal cancer have focused on epithelial carcinoma cells and any contribution of mesenchymal cells to carcinogenesis has received only limited attention. Analysis of surrounding stromal cells should be conducted for a more comprehensive understanding.
It is widely accepted that accumulation of genetic alterations is involved in transition from the normal mucosa to adenocarcinomas via adenomas in the colorectum. APC mutation and loss, K‐ras mutation, Smad2/4 loss, and TP53 mutation and loss are well documented,5,6,7,8 along with altered DNA methylation with progression to carcinoma.9 In colorectal carcinomas, two types of genetic instability, microsatellite instability (MSI), and chromosomal instability (CIN) are frequently detected. MSI is characterised by accumulation of somatic alterations in simple repeated nucleotide sequences called microsatellites10,11 while CIN is characterised by loss of heterozygosity (LOH) at sites of a number of cancer related genes.12,13 The precise mechanisms leading to CIN are not well understood. In sporadic colorectal carcinomas (S‐CRC), it has been reported that approximately 15% of tumours demonstrate MSI,14 approximately 50% exhibiting LOH.15
Previously, using a combination of microdissection and genetic instability analysis with microsatellite markers recommended by the National Cancer Institute (NCI), as well as others close to the p53 and BRCA1 genes on chromosome 17(Chr17), we revealed that not only epithelial but also stromal elements demonstrate genetic instability in invasive colorectal carcinomas and that such stromal alteration might influence the genesis of S‐CRCs.16 In addition, Chr17 markers were found to be more sensitive than NCI markers, especially for detecting stromal genetic instability. The adenoma‐carcinoma sequence is accepted as playing a major role in colorectal carcinogenesis, and colorectal adenomas can be regarded as precancerous.6 In addition, MSI was reported to be present in 13% of colorectal tubular adenomas, 53% of serrated adenomas, and 27% of hyperplastic polyps in one series.17
In the present study, using a laser capture microdissection method, we examined genetic instability in both epithelial and stromal cells of tubular adenomas in the colorectum, and analysed possible correlations with histological progression. For this purpose, a combination of Chr17 and NCI standard microsatellite markers was applied to increase sensitivity.
Materials and methods
Samples
A total of 123 sporadic colorectal polyps and 41 sporadic colorectal invasive carcinomas were examined in this study. Specimens were obtained by either endoscopic polypectomy or surgical resection. More than two expert pathologists histologically diagnosed the hyperplastic polyps (HP), tubular adenomas with low grade (TA‐L) or high grade (TA‐H) dysplasia, and adenocarcinomas (Ca) independently.18 Samples of histologically normal mucosa from each patient were obtained for comparison as controls for the genetic instability analysis. Additional controls were provided by 10 normal mucosa specimens from colorectums without tumours surgically resected from diverticulosis patients. Tissues were formalin fixed and paraffin embedded, and serial 3 μm thick sections were cut for haematoxylin‐eosin staining and immunohistochemistry, along with serial 10 μm thick sections for microdissection. For the latter, deparaffinised sections were used after nuclear staining (Histogene LCM Frozen Section Staining Solution; Arcturus, Mountain View, California, USA).
DNA extraction
Microdissection and DNA extraction were carried out as described previously.16,19 Briefly, epithelial and adjacent stroma tissues in the lamina propria were carefully microdissected with a laser capture microdissection system (LM200 and Pixcell IIe; Arcturus) taking care to avoid cross contamination. Normal control DNA for each case was obtained from histologically normal mucosa sufficiently distant from the tumour. The resulting DNA samples were incubated overnight at 65°C in lysis buffer containing proteinase K (PicoPure DNA extraction kit; Arcturus) and applied for polymerase chain reaction (PCR) analysis after heat inactivation of proteinase K at 95°C for 10 minutes.
Microsatellite analysis
PCR was performed for four microsatellite markers selected for analysing allelic instability on Chr17 (dinucleotide repeats), D17S796, TP53, D17S786 (close to the p53 gene),20 and D17S579 (close to BRCA1),21 along with five NCI standard markers for S‐CRC analysis, BAT25, BAT26 (mononucleotide repeats), D2S123, D3S346, and D17S250 (dinucleotide repeats),22 and in chromosome 1, MYCL (tetranucleotide repeat),23 using fluorescent primer pairs (Applied Biosystems, Foster City, California, USA). Genomic instability was detected with the aid of an ABI Prism 310 or ABI Prism 3100 Avant Genetic Analyser, and analysed with GeneScan Analysis and Genotyper software (version 3.7; PE Applied Biosystems). MSI or LOH positivity was determined compared with control DNA (fig 1B).
Figure 1 (A) Representative results of laser capture microdissection analysis and GeneScan (MYCL) examination of cancer tissue. After a tumour lesion was confirmed by examination of haematoxylin‐eosin stained sections, tumour epithelium and surrounding stromal cells were dissected from serial sections of paraffin embedded tissue. Original magnification: ×40. (B) Representative GeneScan findings for epithelium (E) and stroma (S) from adenomas. N, Normal epithelium or stroma.
Immunohistochemistry
Monoclonal anti‐p53 (DO7, ×300 dilution; Novocastra Lab, Newcastle, UK) and polyclonal anti‐Ki67 (MIB1, ×100 dilution, Dako, Glostrup, Denmark) antibodies were used for immunohistochemistry. Deparaffinised histological sections (3 μm thick) from paraffin embedded tissue specimens and frozen sections were cut for immunohistochemistry, conducted using the streptavidin‐biotin peroxidase complex method. LSAB2 (DakoCytometion, Glostrup, Denmark) and Envision Plus (DakoCytometion) kits were applied with slight modification in our laboratory,24 after microwave oven heating (500 W for five minutes, three times, in 10 mM citrate buffer, pH 6.0) to retrieve antigenic activity. Endogenous peroxidase was inhibited by incubation with 0.3% H2O2 in methanol for 30 minutes. Then, histological sections were incubated with the first antibodies overnight at 4°C and then the second antibodies and substrate. Faint nuclear counterstaining was finally achieved with 5% methyl green. Labelling indices (LI) for p53 and Ki‐67 were calculated as percentages of nuclear positive cells in 1000 cells counted.
Statistical analysis
Differences in distributions between variables were calculated using the χ2 test or Fisher's exact test, as appropriate. Probability values <0.05 were considered significant.
Ethics
This work using pathological samples in Kitasato University and Kitasato University East Hospitals was approved by our Medical School and University Hospital Ethics Committee.
Results
Clinicopathological findings for the colorectal lesions are summarised in table 1. Results for genetic instability are summarised in table 2. No genetic instability was detected in control mucosa specimens.
Table 1 Clinicopathological features of colorectal tumours.
| Clinicopathological factor | Normal control (%) | HP (%) | TA‐L (%) | TA‐H (%) | Ca (%) |
|---|---|---|---|---|---|
| Total No | 10 | 34 | 38 | 51 | 41 |
| Age (y)* | 51.5 (12.6) | 61.0 (12.6) | 68.8 (10.5) | 66.4 (10.4) | 68.2 (9.9) |
| Tumour size (mm)* | 5.7 (3.0) | 6.1 (4.4) | 10.0 (5.9) | 41.4 (15.9) | |
| Sex | |||||
| Male | 7 (70.0) | 25 (73.5) | 28 (73.7) | 39 (76.5) | 28 (68.3) |
| Female | 3 (30.0) | 9 (26.5) | 10 (26.3) | 12 (23.5) | 13 (31.7) |
| Location | |||||
| Proximal | 5 (50.0) | 10 (29.4) | 21 (55.3) | 19 (37.3) | 13 (31.7) |
| Distal | 5 (50.0) | 24 (70.6) | 17 (44.7) | 32 (62.8) | 28 (68.3) |
| Differentiation | |||||
| Well | 18 (43.9) | ||||
| Moderate | 20 (48.8) | ||||
| Poor | 3 (7.3) |
*Values are mean (SD).
HP, hyperplastic polyp; TA‐L, tubular adenoma with low grade dysplasia; TA‐H, tubular adenoma with high grade dysplasia; Ca, adenocarcinoma.
Table 2 Genetic instability of the epithelium and stroma in colorectal tumours.
| MSI+/informative cases (%)* | p Value† | LOH+/informative cases (%)* | p Value† | ||||
|---|---|---|---|---|---|---|---|
| Epithelium | Stroma | Epithelium | Stroma | ||||
| NCI markers | Control | 0/10 (0) | 0/10 (0) | NS | 0/10 (0) | 0/10 (0) | NS |
| HP | 1/34 (2.9)a | 1/34 (2.9) | NS | 0/34 (0)j,k | 0/34 (0) | NS | |
| TA‐L | 2/38 (5.3)b | 0/38 (0) | NS | 3/38 (7.9)l | 0/38 (0)w | NS | |
| TA‐H | 2/51(3.9)c | 1/51 (2.0) | NS | 6/51 (11.8)k | 1/51 (2.0) | NS | |
| CA | 9/41 (22.0)a,b,c | 1/41 (2.4) | 0.0069 | 10/41 (24.4)j,k | 4/41 (9.8)w | NS | |
| Chr17 markers | Control | 0/10 (0) | 0/10 (0) | NS | 0/10 (0)m | 0/10 (0) | NS |
| HP | 1/34 (2.9)d | 0/34 (0) | NS | 1/34 (2.9)n | 1/34 (2.9) | NS | |
| TA‐L | 1/38 (2.6)e | 0/38 (0) | NS | 1/38 (2.6)o,p | 2/38 (5.3) | NS | |
| TA‐H | 5/51 (9.8) | 2/51 (3.9) | NS | 9/51 (17.6)n,p,q | 3/51(5.9) | NS | |
| CA | 8/41 (19.5)d,e | 3/41 (7.3) | NS | 21/41 (51.2)m,o,q | 4/41(9.8) | <0.0001 | |
| Total markers‡ | Control | 0/10 (0)f | 0/10 (0) | NS | 0/10 (0)r | 0/10 (0) | NS |
| HP | 3/34 (8.8)g | 1/34 (2.9) | NS | 1/34 (2.9)s,t | 1/34 (2.9)x | NS | |
| TA‐L | 2/38 (5.3)h | 2/38 (5.3) | NS | 5/38 (13.2)u | 2/38 (5.3) | NS | |
| TA‐H | 7/51(13.7)i | 3/51 (5.9) | NS | 14/51 (27.5)t,v | 3/51 (5.9) | 0.0035 | |
| CA | 14/41 (34.2)f,g,h,i | 3/41 (7.3) | 0.0027 | 25/41 (61.0)r,s,u,v | 7/41 (17.1)x | <0.0001 | |
*Percentage microsatellite instability (MSI) or loss of heterozygosity (LOH)+/informative case.
†Significant difference between epithelium and stroma.
‡Total markers: National Cancer Institute (NCI) markers, chromosome 17 (Chr17) markers, and MYCL.
HP, hyperplastic polyp; TA‐L, tubular adenoma with low grade dysplasia; TA‐H, tubular adenoma with high grade dysplasia; Ca, adenocarcinoma.
a, b, d, e, f, i, k, l, n, p, w, x, p<0.05; c, g, p<0.01; h, j, m, t, v , p<0.005; q, r, p<0.001; o, s, u, p<0.0001.
Genetic instability in tumour epithelium (table 2, fig 2A, B)
Figure 2 Frequencies of genetic instability in colorectal tumours. C, normal mucosa; HP, hyperplastic polyp; TA‐L, tubular adenoma with low grade dysplasia; TA‐H, tubular adenoma with high grade dysplasia; Ca, invasive adenocarcinoma; NCI, National Cancer Institute standard markers; Chr17, chromosome 17 markers. (A) Epithelial loss of heterozygosity (LOH) with NCI and Chr17 markers. (B) Epithelial microsatellite instability (MSI) with NCI and Chr17 markers. (C) Stromal LOH with NCI and Chr17 markers. (D) Stromal MSI with NCI and Chr17 markers. (E) Epithelial LOH and MSI with all markers in combination (NCI and Chr17 markers and MYCL). (F) Stromal LOH and MSI with all markers in combination. Percentages of MSI or LOH positive/informative lesions (%) are shown by the vertical axis. Bars indicate positive for 1, 2, 3, or 4 or more markers. *p<0.05, †p<0.01, ‡p<0.005, §p<0.001, ¶p<0.0001. a, b, d, i, m, p, p<0.05; g, j, p<0.01; f, h, p<0.005; o, p<0.001; c, e, n, p<0.0001.
Frequencies of epithelial LOH with NCI markers were increased stepwise from HP 0%; TA‐L 7.9%; TA‐H 11.8%; and Ca 24.4% (table 2). However, with the Chr17 markers, frequencies were significantly lower in HP and TA‐L than in TA‐H (p<0.05) (fig 2A). LOH in Ca was significantly increased compared with HP and TA‐L using both NCI (HP p<0.005, TA‐L p<0.05) and Chr17 (HP p<0.0001, TA‐L p<0.0001) markers and compared with TA‐H with Chr17 markers (p<0.01) (fig 2A). Considering the numbers of positive markers of LOH, only one was positive with the NCI recommended examples in each lesion, while multiple Chr17 markers showed LOH in TA‐H and Ca (fig 2A). The frequencies of LOH with Chr17 markers were significantly greater than NCI markers, particularly for two or more markers (TA‐H, p<0.01; Ca, p<0.0001) and three or more markers (TA‐H p<0.05; Ca p<0.005) (fig 2A).
Regarding epithelial MSI, the frequencies with NCI markers were significantly higher for Ca than HP and TA‐L categories (HP p<0.05, TA‐L p<0.05).
Genetic instability in tumour stroma (table 2, fig 2C, D)
Frequencies of stromal LOH with NCI markers were 2.0% in TA‐H, 9.8% in Ca, but none in HP and TA‐L. The Ca value was significantly greater than that for TA‐L (p<0.05) (fig 2C). Frequencies of LOH with Chr17 markers gradually increased with progression but without significance (2.9% in HP, 5.3% in TA‐L, 5.9% in TA‐H, and 9.8% in Ca). Frequencies of MSI were very low (fig 3D).
Figure 3 Representative immunohistochemical staining of p53 and Ki‐67. p53 in hyperplastic polyps (HP) (A), in tubular adenomas with low grade dysplasia (TA‐L) (B), in tubular adenomas with high grade dysplasia (TA‐H) (C), and in adenocarcinomas (D). Ki‐67 in HP (E), in TA‐L (F), in TA‐H (G), and in Ca (H). Original magnification: ×100.
Genetic instability with all markers in combination (table 2, fig 2E, F)
With the total of 10 NCI, Chr17, and MYCL markers, frequencies of epithelial LOH tended to be increased with histological progression. Considering the numbers of positive markers, epithelial LOH was detected with multiple markers significantly more frequently in the TA‐H and Ca categories (fig 2E).
Frequencies of epithelial MSI with all markers in combination also tended to increase along with histological progression. Values were significantly higher for Ca than HP (p<0.01), TA‐L (p<0.005), or TA‐H (p<0.05). Considering the numbers of positive markers, LOH was detected with multiple markers in TA‐H and Ca, and MSI was detected with multiple markers in TA‐L, TA‐H, and Ca. Although stromal genetic instability was infrequent, a significant difference was found between Ca and HP LOH (p<0.05).
Relations between status for genetic instability and tumour location (table 3)
Table 3 Relations between genetic instability and tumour location.
| Total | LOH+ | MSI+ | LOH+ and MSI+ | |||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Epithelium | Stroma | Epithelium | Stroma | Epithelium | Stroma | |||||
| HP | Proximal | 10 | 1/10 | 0/10 | 2/10 | 0/10 | 0/10 | 1/10 | ||
| Distal | 24 | 0/24 | 0/24 | 1/24 | 0/24 | 0/24 | 0/24 | |||
| TA‐L | Proximal | 21 | 2/21 | 2/21 | 2/21 | 0/21 | 0/21 | 0/21 | ||
| Distal | 17 | 3/17 | 0/17 | 0/17 | 2/17 | 0/17 | 0/17 | |||
| TA‐H | Proximal | 19 | 4/19 | 2/19 | 3/19 | 2/19 | 2/19 | 0/19 | ||
| Distal | 32 | 8/32 | 1/32 | 2/32 | 1/32 | 0/32 | 0/32 | |||
| Ca | Proximal | 13 | 0/13* | 3/13 | 5/13† | 0/13 | 2/13 | 1/13 | ||
| Distal | 28 | 16/28* | 2/28 | 0/28† | 1/28 | 7/28 | 1/28 | |||
Proximal: caecum, ascending colon, and transverse colon.
Distal: descending colon, sigmoid colon, and rectum.
HP, hyperplastic polyp; TA‐L, tubular adenoma with low grade dysplasia; TA‐H, tubular adenoma with high grade dysplasia; Ca, adenocarcinoma.
*p<0.05; †p<0.005.
Data for genetic instability with reference to tumour location are summarised in table 3. There was no significant link for HP, TA‐L, or TA‐H. However, positivity for MSI in Ca was significantly higher (p<0.005) on the proximal side than on the distal side. Furthermore, the frequency of epithelial LOH in Ca of the distal colon was significantly higher (p<0.005) than that on the proximal side. No significant correlations between stromal genetic instability and tumour location were found.
Relation between p53 overexpression, cell proliferation activity (Ki‐67 LI), and epithelial genetic instability
As the Chr17 markers are in the vicinity of the p53 gene, the relation between epithelial genetic instability and p53 protein expression was examined by immunohistochemistry, representative findings being shown in fig 3. In HP and TA‐H, p53 protein expression was significantly greater in LOH positive lesions than in those without genetic instability (HP p<0.005, TA‐H p<0.005) (fig 4A), but no variation in cell proliferation, as assessed by Ki‐67, was apparent (fig 4B). No significant correlation between MSI positivity and p53 overexpression was found.
Figure 4 (A) p53 overexpression with reference to the genetic instability status of colorectal tumour epithelium. (B) Ki‐67 expression with reference to the genetic instability status of colorectal tumour epithelium. HP, hyperplastic polyp; TA‐L, tubular adenoma with low grade dysplasia; TA‐H, tubular adenoma with high grade dysplasia; Ca, adenocarcinoma. LOH, loss of heterozygosity; MSI, microsatellite instability. Values are mean (SD).
Discussion
Participation of genetic instability in colorectal carcinoma development has been indicated by many reports of LOH and MSI.25,26 The fact that MSI exists in 10% of colorectal adenomas27 may imply a role in the adenoma‐carcinoma sequence and the present study provided concrete evidence that this is indeed the case. Previously, we revealed that not only epithelium but also stroma may demonstrate genetic instability in sporadic invasive colorectal carcinomas.16 Furthermore, in an ulcerative colitis associated tumour series, representative of the established chronic inflammation‐carcinoma sequence,28,29 we found both epithelial and stromal genetic instability in regenerative mucosa, dysplasia, and adenocarcinoma, suggesting an influence of the stromal microenvironment on epithelial cells from an early stage in the development of neoplasms.19
In the present study, we further demonstrated genetic instability in epithelial and stromal cells, and analysed their correlation with histological progression of colorectal tumours. In a previous report,17 the prevalence of epithelial genetic instability varied between tubular adenoma (MSI in 13%) and serrated adenoma (MSI in 57%). Therefore, we focused on tubular adenomas because villous and serrated adenomas may possess different biological and genetic features.9 We found that epithelial genetic instability increases with progression of histological lesions, whereas LOH and MSI in stromal elements tend to be more constant in TA‐L, TA‐H, and invasive carcinomas. In line with the finding of frequent genetic changes in mammary stromal tissue in breast carcinoma patients,30 and the demonstration of conversion of colonic adenomas to adenocarcinomas in nude mice with inflammation associated stroma,27 the results of this study suggest that early changes in the stroma may be associated with epithelial tumorigenesis. When MSI was categorised into high level (MSI‐H; MSI found in four or more markers) and low level (MSI‐L; MSI found in 1–3 markers) MSI, MSI‐H was found to exist in TA‐H and Ca epithelium (fig 2E) and not in stroma (fig 2F). Although MSI‐H, defects of mismatch repair enzymes, and MSI in mononucleotide markers such as BAT‐25 or BAT‐26 are considered to be correlated,25,26 some stromal lesions with MSI‐L showed major shift bands in mononucleotide markers (fig 1B, BAT‐26). The relations between the major shift bands in stroma and mismatch repair enzymes remain to be analysed. Thus the significance of MSI‐L found in stroma should be clarified in relation to tumorigenesis.
Previously, we reported that cell proliferation estimated by Ki‐67 LI increased stepwise from TA‐L and TA‐H through to invasive carcinomas.31 In this study, however, we did not find significant differences in Ki‐67 LI among lesions with or without genetic instability. Furthermore, our immunohistochemical investigation showed p53 overexpression was significantly greater in LOH positive than LOH negative TA‐H. Some cases of TA‐H showed diffuse p53 staining which might indicate a mutation in the tumour suppressor gene although we could not confirm this here. However, other TA‐H showed scattered expression of p53 with a higher LI than TA‐L (data not shown). Therefore, the increased p53 LI in TA‐H with LOH may be due to the coexistence of wild‐type and mutant p53 components.
Previously, evidence was presented that MSI‐H colorectal tumours are more likely to be right sided (proximal) than their MSI‐L or MSI stable counterparts.14,32 The present study also demonstrated epithelial MSI predominantly in carcinomas of the proximal colon. In addition, all epithelial LOH positive carcinomas were located in the distal colon, suggesting differences in carcinogenesis between the two sites.33 To our knowledge, there have hitherto been no reports of significant correlations between LOH and tumour location. As the number of tumours analysed was rather small in the present study, this point needs further investigation with a larger series.
In conclusion, the present results suggest that stromal genetic instability is associated with colorectal adenoma development. Contrary to the general belief that abnormalities in stroma occur as reactions to epithelial tumour cells, our data support the hypothesis that an alternative pathway with prior influence of stromal genetic instability on epithelial cells may exist in the adenoma‐adenocarcinoma sequence. Further studies are warranted to test this hypothesis.
Acknowledgements
This work was partly supported by Grants‐in‐Aid for Scientific Research from Kitasato University Graduate School of Medical Sciences; the Japanese Ministry of Education, Culture, Sports, Science and Technology (No 1377094, No 14570160, No 16590293); and the Uehara Memorial Foundation.
Abbreviations
Ca - adenocarcinoma
Chr17 - chromosome 17
CIN - chromosomal instability
HP - hyperplastic polyp
LI - labelling index
LOH - loss of heterozygosity
MSI - microsatellite instability
MSI‐H - high level MSI
MSI‐L - low level MSI
NCI - National Cancer Institute
PCR - polymerase chain reaction
S‐CRC - sporadic colorectal carcinoma
TA‐H - tubular adenoma with high grade dysplasia
TA‐L - tubular adenoma with low grade dysplasia
Footnotes
Conflict of interest: None declared.
References
- 1.Marsh M N, Trier J S. Morphology and cell proliferation of subepithelial fibroblasts in adult mouse jejunum. II. Radioautographic studies. Gastroenterology 197467636–645. [PubMed] [Google Scholar]
- 2.Shekhar M P, Werdell J, Santner S J.et al Breast stroma plays a dominant regulatory role in breast epithelial growth and differentiation: implications for tumor development and progression. Cancer Res 2001611320–1326. [PubMed] [Google Scholar]
- 3.Camps J L, Chang S M, Hsu T C.et al Fibroblast‐mediated acceleration of human epithelial tumor growth in vivo. Proc Natl Acad Sci U S A 19908775–79. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Hom Y K, Young P, Wiesen J F.et al Uterine and vaginal organ growth requires epidermal growth factor receptor signaling from stroma. Endocrinology 1998139913–921. [DOI] [PubMed] [Google Scholar]
- 5.Morson B C. Evolution of cancer of the colon and rectum. Cancer 197434(suppl)845–849. [DOI] [PubMed] [Google Scholar]
- 6.Vogelstein B, Fearon E R, Hamilton S R.et al Genetic alterations during colorectal‐tumor development. N Engl J Med 1988319525–532. [DOI] [PubMed] [Google Scholar]
- 7.Fearon E R, Vogelstein B. A genetic model for colorectal tumorigenesis. Cell 199061759–767. [DOI] [PubMed] [Google Scholar]
- 8.Kinzler K W, Vogelstein B. Lessons from hereditary colorectal cancer. Cell 199687159–170. [DOI] [PubMed] [Google Scholar]
- 9.Jass J R. Serrated route to colorectal cancer: back street or super highway? J Pathol 2001193283–285. [DOI] [PubMed] [Google Scholar]
- 10.Thibodeau S N, Bren G, Schaid D. Microsatellite instability in cancer of the proximal colon. Science 1993260816–819. [DOI] [PubMed] [Google Scholar]
- 11.Jass J R, Do K A, Simms L A.et al Morphology of sporadic colorectal cancer with DNA replication errors. Gut 199842673–679. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Lengauer C, Kinzler K W, Vogelstein B. Genetic instability in colorectal cancers. Nature 1997386623–627. [DOI] [PubMed] [Google Scholar]
- 13.Lengauer C, Kinzler K W, Vogelstein B. Genetic instabilities in human cancers. Nature 1998396643–649. [DOI] [PubMed] [Google Scholar]
- 14.Michael‐Robinson J M, Biemer‐Huttmann A, Purdie D M.et al Tumour infiltrating lymphocytes and apoptosis are independent features in colorectal cancer stratified according to microsatellite instability status. Gut 200148360–366. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Goel A, Arnold C N, Niedzwiecki D.et al Characterization of sporadic colon cancer by patterns of genomic instability. Cancer Res 2003631608–1614. [PubMed] [Google Scholar]
- 16.Matsumoto N, Yoshida T, Yamashita K.et al Possible alternative carcinogenesis pathway featuring microsatellite instability in colorectal cancer stroma. Br J Cancer 200389707–712. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Iino H, Jass J R, Simms L A.et al DNA microsatellite instability in hyperplastic polyps, serrated adenomas, and mixed polyps: a mild mutator pathway for colorectal cancer? J Clin Pathol 1999525–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Jass J R, Sobin L. H. Histological typing of intestinal tumours. In, Sobin LH, ed. WHO international histological classification of tumours, 2nd Edn Berlin, Springer Verlag 1989
- 19.Matsumoto N, Yoshida T, Okayasu I. High epithelial and stromal genetic instability of chromosome 17 in ulcerative colitis‐associated carcinogenesis. Cancer Res 2003636158–6161. [PubMed] [Google Scholar]
- 20.Gyapay G, Morissette J, Vignal A.et al The 1993–94 Genethon human genetic linkage map. Nat Genet 19947246–339. [DOI] [PubMed] [Google Scholar]
- 21.Anderson L A, Friedman L, Osborne‐Lawrence S.et al High‐density genetic map of the BRCA1 region of chromosome 17q12–q21. Genomics 199317618–623. [DOI] [PubMed] [Google Scholar]
- 22.Boland C R, Thibodeau S N, Hamilton S R.et al A National Cancer Institute Workshop on Microsatellite Instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer. Cancer Res 1998585248–5257. [PubMed] [Google Scholar]
- 23.Souza R F, Lei J, Yin J.et al A transforming growth factor beta 1 receptor type II mutation in ulcerative colitis‐associated neoplasms. Gastroenterology 199711240–45. [DOI] [PubMed] [Google Scholar]
- 24.Yoshida T, Mikami T, Mitomi H.et al Diverse p53 alterations in ulcerative colitis‐associated low‐grade dysplasia: full‐length gene sequencing in microdissected single crypts. J Pathol 2003199166–175. [DOI] [PubMed] [Google Scholar]
- 25.Young J, Simms L A, Biden K G.et al Features of colorectal cancers with high‐level microsatellite instability occurring in familial and sporadic settings: parallel pathways of tumorigenesis. Am J Pathol 20011592107–2116. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Duval A, Hamelin R. Mutations at coding repeat sequences in mismatch repair‐deficient human cancers: toward a new concept of target genes for instability. Cancer Res 2002622447–2454. [PubMed] [Google Scholar]
- 27.Okada F, Kawaguchi T, Habelhah H.et al Conversion of human colonic adenoma cells to adenocarcinoma cells through inflammation in nude mice. Lab Invest 2000801617–1628. [DOI] [PubMed] [Google Scholar]
- 28.Collins R H, Jr, Feldman M, Fordtran J S. Colon cancer, dysplasia, and surveillance in patients with ulcerative colitis. A critical review. N Engl J Med 19873161654–1658. [DOI] [PubMed] [Google Scholar]
- 29.Okayasu I, Hana K, Yoshida T.et al Significant increase of colonic mutated crypts in ulcerative colitis correlatively with duration of illness. Cancer Res 2002622236–2238. [PubMed] [Google Scholar]
- 30.Moinfar F, Man Y G, Arnould L.et al Concurrent and independent genetic alterations in the stromal and epithelial cells of mammary carcinoma: implications for tumorigenesis. Cancer Res 2000602562–2566. [PubMed] [Google Scholar]
- 31.Mikami T, Yoshida T, Akino F.et al Apoptosis regulation differs between ulcerative colitis‐associated and sporadic colonic tumors. Association with survivin and bcl‐2. Am J Clin Pathol 2003119723–730. [DOI] [PubMed] [Google Scholar]
- 32.Ward R, Meagher A, Tomlinson I.et al Microsatellite instability and the clinicopathological features of sporadic colorectal cancer. Gut 200148821–829. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33.Nagengast F M, van der Werf S D, Lamers H L.et al Influence of age, intestinal transit time, and dietary composition on fecal bile acid profiles in healthy subjects. Dig Dis Sci 198833673–678. [DOI] [PubMed] [Google Scholar]