Int. J. Cancer: 119, 2513–2519 (2006) ' 2006 Wiley-Liss, Inc.
Heterogeneous microsatellite instability observed within epithelium of ulcerative colitis Kazuhide Ozaki1, Takeshi Nagasaka1, Kenji Notohara2, Takeshi Kambara1, Masanori Takeda1, Hiromi Sasamoto1, Jeremy R. Jass3, Noriaki Tanaka1 and Nagahide Matsubara1* 1 Department of Gastroenterological Surgery and Surgical Oncology, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan 2 Department of Pathological Research, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan 3 Department of Pathology, McGill University, Montreal, QC, Canada Microsatellite instability (MSI) has been associated with colitic cancer. However, reported frequency of MSI was varied and the association of MSI with mismatch repair (MMR) deficiency was unclear. In addition, the occurrence of genetic alterations in stromal cells within ulcerative colitis (UC) is still controversial. We therefore sampled 164 microareas in various pathological lesions of UC with or without colitic cancer and studied the MSI status in relation to the DNA repair protein expressions. A total of 129 microfoci from colorectal tissue of 5 colitic cancer patients and 35 microfoci of 7 UC patients (without neoplasm) were carefully sampled by laser-capture microdissection. MSI was analyzed in each microsamples. The protein expression of MMR genes (MLH1, MSH2, MSH6), O6-methylguanine-DNA methyltransferase and p53 were assessed by immunohistochemical analysis. Variety of di-nulcleotide microsatellite markers was altered in individual microfoci from different morphological epithelial lesions, in full range of nonneoplastic epithelium to colitic cancer. Interestingly, MSI was not observed in stromal cells at any sites, including those within colitic cancer/dysplasia lesions. Expression of the MMR proteins was not lost in any of the lesions examined. Microsatellite alterations rather seem to be related to the initiation than to the progression of colitic cancer. ' 2006 Wiley-Liss, Inc. Key words: colitic cancer; ulcerative colitis; microsatellite instability; stroma; mismatch repair
Colorectal epithelium with a long-standing ulcerative colitis (UC) has an increased risk of developing cancer.1–6 An invasive colitic cancer often arises from a preceding flat dysplastic epithelium and tend to develop multifocally over the background inflamed epithelium, whilst sporadic colorectal cancer (CRC) develops from a preexisting adenoma, suggesting a dissimilar carcinogenic pathway in colitic cancer.7–15 In lesions undergoing chronic inflammation, microsatellite instability (MSI) is a possible genetic alteration, and high- and low-level of MSI (MSI-H and MSI-L) has been described in UC associated neoplasm (UCAN).16–23 However, the reported frequency of MSI-H, a consequence of a defective mismatch repair (MMR) system (notably MLH1 or MSH2), and MSI-L, a consequence in part of O6-methylguanine-DNA methyltransferase (MGMT) deficiency,24 has varied considerably from 9 to 50%, and 11 to 85%, respectively.22,25–27 In situation of considerable genetic heterogeneity within a sample, collection of samples without microdissection could possibly obscure the findings of MSI.28,29 In fact, analysis of mixed MSI-L components could result in a false diagnosis as MSI-H. To verify true MSI status and to clarify the significance of MSI in colitic carcinogenesis, precise analysis of individual microfoci from various pathological lesions (including stroma) is required.16,18,19 Accordingly, we sampled 164 microfoci, including nondysplastic epithelium, dysplastic epithelium and cancers from colorectal specimens from UC patients to assess the precise (predicted heterogeneous) genetic alterations that may occur in colitic cancer. We further analyzed the protein expression of MLH1, MSH2, MSH6, MGMT and P53. Finally we investigated genetic alterations in stroma cells within cancer/dysplastic epithelium, because possible genetic alterations within stromal components in UC carcinogenesis were reported.30 Publication of the International Union Against Cancer
Material and methods Sample collection and microdissection All tissue samples were obtained from the surgical specimens from 5 UC patients with developed UCAN and from 7 UC patients without neoplasm. The patients underwent surgery at our institution or our affiliated hospitals from 1998 to 2000. Multiple microareas of nonneoplastic epithelium, dysplastic epithelium and cancer were obtained using a PixCel laser capture microscope (LM100; Olympus, Tokyo, Japan) with an infrared diode laser (Arcturus Engineering, CA). A total of 164 microepithelial samples from 12 UC patients and 129 microsamples from 5 UCAN patients were collected and separately analyzed. We extracted and analyzed DNAs from 49 microfoci of nonneoplastic epithelium, 48 microfoci of 5 dysplastic lesions, and 32 microfoci of 4 cancer lesions derived from the 5 UCAN patients. We also extracted DNA from 35 microsamples of different nonneoplastic epithelium derived from the 7 UC without cancer (patient nos. 6–12). Additionally, 28 microloci from stromal areas were collected from 5 UCAN patients. Stromal microsamples within dysplastic, and nonneoplastic areas were carefully microdissected to avoid contamination of epithelial-origin cells, however, some infiltrated lymphocytes were included in the microsamples. As for cancer, stromal cells at the peripheral part of cancer were carefully collected to avoid contamination of cancer cells. Normal control tissue was collected from colonic nonstriated muscle of each specimen. Our study received approval from the institutional review board of the faculty of medicine of Okayama University, Japan. For each sample, serial sections were cut from formalin-fixed paraffin blocks and the first section was counter-stained with hematoxylin and eosin (H&E) for histological diagnosis. The lesions were classified according to the Inflammatory Bowel Disease-Dysplasia Morphology Study Group Criteria (IBDDMSGC) published in 1983.11 The histological diagnosis was conducted by a gastrointestinal pathologist (K. N). To maximize the purity of the target epithelial cells, we applied laser capture microdissection (LCM) technique. Tissue was transferred in a thermal polymer disk from 5 to 8 lm deparaffinized unstained serial slides by using a laser beam. This method enabled us to enrich the cellularity of the collected epithelium, at a percentage of 75 or greater. One microsample was dissected by 50–60 shots of laser beam and incubated in 20 lm of lysis buffer [20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.5% Tween 20, 200 lg/ml proteinase K] for 24 hr at 37°C, then for 15 min at 95°C to inactivate proteinase K.
Grant sponsor: Japanese Ministry of Education, Science, Sports and Culture; Grant numbers: 17390368, 17591403, 17591402, 14031227. *Correspondence to: Department of Gastroenterological Surgery and Surgical Oncology, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700 8558, Japan. Fax: 181-86-221-8775. E-mail:
[email protected] Received 1 December 2005; Accepted 18 April 2006 DOI 10.1002/ijc.22095 Published online 23 August 2006 in Wiley InterScience (www.interscience. wiley.com).
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FIGURE 1 – Laser captured microdissection of various colorectal epithelia from ulcerative colitis patients. With the guide of H&E stained slices (a), dysplastic regions were laser-captured from nonstained slices under microscopy. (b) Unstained slice before capture, (c) unstained slice after dysplastic regions were removed and (d) captured unstained dysplastic regions. Figures (e)–(h) indicate the case of invasive colitic cancer tissues. (e) H&E stained slices, (f) unstained slice before capture, (g) unstained slice after cancer regions were removed and (h) captured unstained cancer regions.
Microsatellite analysis The MSI testing for each tumor was determined on the basis of an examination of 12 microsatellite markers (BAT25, BAT26, BAT40, D2S123, D5S107, D5S346, D8S87, D17S261, D17S250, D18S35, D18S58 and MYCL1) described previously.31 BAT25, BAT26 and BAT40 are mono-nucleotide repeat markers and MYCL1 is the tetra-repeat markers. The rest of the markers are dinucleotide repeat ones. We classified tumors as MSI-L and MSIH, if 0%,
