This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Save to my personal folders Download to citation manager Rights & Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Svrcek, M. Articles by Fléjou, J.-F. Search for Related Content PubMed PubMed Citation Articles by Svrcek, M. Articles by Fléjou, J.-F. Social Bookmarking                            What's this?

Specific Clinical and Biological Features Characterize Inflammatory Bowel Disease–Associated Colorectal Cancers Showing Microsatellite Instability

Magali Svrcek, Jamila El-Bchiri, Alexandra Chalastanis, Emilie Capel, Sylvie Dumont, Olivier Buhard, Carla Oliveira, Raquel Seruca, Céline Bossard, Jean-François Mosnier, Françoise Berger, Emmanuelle Leteurtre, Anne Lavergne-Slove, Marie-Pierre Chenard, Richard Hamelin, Jacques Cosnes, Laurent Beaugerie, Emmanuel Tiret, Alex Duval, Jean-François Fléjou

From the Assistance Publique-Hôpitaux de Paris (AP-HP), Hôpital Saint-Antoine, Service d'Anatomie Pathologique; Université Pierre et Marie Curie-Paris; Institut National de la Santé et de la Recherche Médicale, U762, Centre d’Etude du Polymorphisme Humain; AP-HP, Hôpital Lariboisière, Service central d'Anatomie et Cytologie Pathologiques; AP-HP, Hôpital Saint-Antoine, Service de Gastroentérologie et Nutrition; AP-HP, Hôpital Saint-Antoine, Service de Chirurgie Viscérale, Paris; Service d'Anatomie Pathologique, Hôpital Hôtel-Dieu, Nantes; Service d'Anatomie Pathologique, Centre Hospitalier Lyon Sud, Lyon; Service d'Anatomie Pathologique, Centre Hospitalier Régional et Universitaire de Lille, Pôle Biologie-Pathologie-Parc Eurosanté, Lille; Service d'Anatomie Pathologique Générale, Centre Hospitalier Universitaire de Hautepierre, Strasbourg, France; and Instituto de Patologia e Immunologia Molecular da Universidade do Porto, Porto, Portugal

Address reprint requests to Magali Svrcek, MD, Service d'Anatomie et Cytologie Pathologiques, Hôpital Saint-Antoine, 184, rue du Faubourg Saint-Antoine, F75571 Paris cedex 12, Paris, France; e-mail: magali.svrcek{at}sat.aphp.fr

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS Appendix REFERENCES

 
Purpose Microsatellite instability (MSI) due to mismatch repair (MMR) deficiency has been reported to occur at variable frequencies in inflammatory bowel disease–associated intestinal neoplasias (IBD-Ns). We investigated a large series of IBD-N for associations between MSI and several biologic and clinical parameters related to tumors, patients, and their treatment.

Patients and Methods A total of 277 IBD-Ns in 205 patients were screened for MSI. Biologic and clinical variables of patients with high levels of DNA microsatellite instability high (MSI-H) were collected and compared with those associated with 33 MSI-H non-IBD colorectal cancers (CRCs).

Results A total of 27 IBD-Ns from 17 patients were found to be MSI-H. Compared with sporadic MSI-H CRCs, patients presented with a younger age at diagnosis, and there was no female predominance and no right-sided predominance. Unlike sporadic MSI-H CRCs, MSI-H IBD-Ns presented with heterogeneous mismatch repair defects involving MLH1, MSH2, MSH6, or PMS2, and a low frequency of MLH1 promoter methylation. They exhibited frequent BRAF mutations and frameshift mutations in genes containing coding repeat sequences.

Conclusion The mechanisms underlying MMR deficiency in MSI-H IBD-Ns are different from those in sporadic MSI-H tumors and seem to be more related to those observed in hereditary MSI-H tumors. However, BRAF mutations were observed in MSI-H IBD-Ns, similar to sporadic MSI-H tumors, but unlike hereditary MSI-H tumors. Finally, the mutational events in target genes for instability are the same in MSI-H IBD-N tumors as in non-IBD sporadic and hereditary colorectal MSI-H cancers, indicating a colon-related repertoire of target gene alterations.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS Appendix REFERENCES

 
The microsatellite instability high (MSI-H) phenotype is associated with the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome. It is also observed in approximately 10% to 15% of sporadic colorectal, gastric, and endometrial cancers.^1-3 MSI-H colorectal cancers (CRCs) are characterized by particular morphologic features, including frequent location in the proximal colon, poor tumor differentiation, mucinous or signet ring cell pattern, prominent tumor-infiltrating lymphocytes, marked Crohn's-like lymphoid reaction, and expanding margins of invasion.⁴ In tumor samples, the MSI-H phenotype can be determined by polymerase chain reaction (PCR) according to international criteria.⁵ It is correlated with the loss of mismatch repair (MMR) protein expression affecting MLH1, MSH2, MSH6, or PMS2 by immunohistochemical study.⁶ Whereas MSI-H CRCs associated with HNPCC patients are characterized by heterogeneous MMR defects, MMR deficiency in the great majority of MSI-H sporadic CRCs is due to methylation-induced transcriptional silencing of the MLH1 gene.⁷ In MSI-H CRCs, a high incidence of mutually exclusive somatic mutations of either BRAF or KRAS has been described.^8-10 More specifically, HNPCC-related tumors present only with KRAS mutation, whereas BRAF mutations occur almost exclusively in MSI-H sporadic CRCs showing methylation of the MLH1 MMR gene.^11-13 Finally, all MSI-H CRCs show a characteristic mutator phenotype, characterized by frequent frameshift mutations that occur in so-called target genes for MSI that contain coding microsatellite sequences.¹⁴

In a previous study, we showed that the mutator pathway was a feature of immunodeficiency and chronic inflammation-related post-transplantation non-Hodgkin's lymphomas.¹⁵ Intestinal cancers developing in patients suffering from inflammatory bowel disease (IBD; eg, ulcerative colitis [UC] and Crohn's disease [CD]), represent another model of carcinogenesis that could be related to chronic inflammation and/or deficient immune status. MSI previously has been reported to be a feature of some IBD-associated intestinal cancers, with variable frequencies ranging from less than 1% to 45%.^16-23 As in MSI-H sporadic CRCs from the general population, MMR deficiency characterizing MSI-H IBD-neoplasm (IBD-N) has been proposed to be due mainly to methylation-induced silencing of MLH1.^21-23 However, to date, an extensive analysis of MMR protein expression has not been reported in a large series of MSI-H IBD-Ns. The frequency of KRAS mutations was found to be lower in UC-associated neoplasias than in sporadic CRCs.^19,24,25 BRAF mutations have been reported in 9% of IBD-associated CRCs.²² Finally, Schulmann et al²³ recently showed significant differences between MSI-H IBD-Ns and MSI-H sporadic CRCs regarding the profile of target gene frameshift mutations at coding microsatellite repeats.

We investigated the presence of MSI in a large series of IBD-Ns. We conducted an extensive and systematic analysis of both primary and secondary mutational events characterizing MSI-H carcinogenesis and evaluated the clinical features associated with MSI-H IBD-Ns in comparison to those seen in either hereditary or sporadic MSI-H non–IBD-associated CRCs.

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS Appendix REFERENCES

 
Patients and Tumor Samples
Two hundred seventy-seven formalin-fixed (n = 248) or Bouin-fixed (n = 29), paraffin-embedded samples from 205 patients with IBD-N (defined as adenocarcinoma, dysplasia, or sporadic adenoma) were used. Nineteen institutions were involved in this retrospective study. Ethics approval was obtained from the Human Research Ethics Committee of the Saint-Antoine Hospital (Paris, France). Information about family history of cancer was obtained from the clinical charts to identify patients fulfilling the Amsterdam criteria or extended Bethesda guidelines for HNPCC.²⁶ Data on medication for IBD were also collected, in particular immunosuppressive drugs (corticosteroids, cyclosporine, azathioprine, or infliximab) and 5-aminosalicyclic acid therapy. The clinical characteristics, the immunohistochemical pattern of MMR protein expression, and the frequencies of MLH1 promoter methylation and BRAF mutations were compared with those observed in a recently reported series of 33 non-IBD MSI-H tumors, of which 18 showed MLH1 promoter methylation and 15 did not. These latter tumors were considered as likely sporadic and likely HNPCC, respectively.²⁷

MSI Testing With Pentaplex PCR
DNA was extracted from formalin-fixed, paraffin-embedded neoplastic tissue, macrodissected with reference to the hematoxylin and eosin–stained section. DNA was extracted after deparaffinization by treatment with sodium dodecyl sulfate–proteinase K and phenol-chloroform-isoamyl alcohol.²⁸ To evaluate MSI, the pentaplex PCR, comprising five quasimonomorphic mononucleotide repeats (BAT-25, BAT-26, NR-21, NR-24, and NR-27) was used.²⁹ Given that the date of fixation, treatment, and conservation were highly variable due to the different origins of paraffin-embedded tissues, it was not always possible to amplify all markers, particularly the longer ones. Samples with results from less than three markers were thus considered as not interpretable. For the other samples, tumors with instability at three or more markers were defined as MSI-H, whereas those with instability at one or two repeats or showing no instability were defined as microsatellite stable (MSS).

Construction and Processing of Tissue Microarrays and Immunohistochemistry
The same paraffin blocks were used for DNA extractions and for immunohistochemistry (IHC) analyses. For tissue microarrays, three core biopsies of 0.6 mm in diameter were taken for each neoplastic lesion (except for very small lesions). The expression of MLH1, MSH2, MSH6, and PMS2 was assessed as described previously.³⁰ Briefly, 4-µm tissue sections were stained with antibody to MLH1 (clone G168-728, 1:70 dilution; PharMingen, San Diego, CA), MSH2 (clone FE11, 1:100 dilution; Calbiochem, Cambridge, MA), MSH6 (clone 44, 1:100 dilution; Becton Dickinson, Lexington, NC), and PMS2 (clone A16-4, 1:150 dilution; BD PharMingen, Le Pont de Claix, France). Samples showing loss of protein expression on tissue microarrays were also tested on whole-section slides.

Histopathology
Clinicopathologic variables were reviewed in IBD-N samples. In each patient sample, the anatomic distribution, degree of differentiation, histologic type,³¹ peritumoral chronic inflammation with or without Crohn's-like lymphoid nodules (present, absent), margins (infiltrating or expanding), and the presence of tumor-infiltrating lymphocytes (classified semiquantitatively as none, discrete, or intense) were noted.⁴ The histologic classification of dysplasia was based on the Vienna classification of GI epithelial neoplasia.³²

BRAF and KRAS Mutations Screening in MSI-H IBD-Ns
Mutational analysis of BRAF was performed by single-stranded conformation polymorphism and heteroduplex analysis, as reported previously.¹⁰ Mutational analysis of KRAS was performed by denaturing gradient gel electrophoresis.

Determination of MLH1 Promoter Methylation
The DNA methylation pattern of the MLH1 promoter region was determined by methylation-specific PCR on bisulfite-treated DNA (1 µg)³³ with primer sequences used by Park et al.³⁴ PCR products from methylated and unmethylated sequences were electrophoresed on a 2.5% agarose gel and visualized by ethidium bromide staining.

Mutation Analysis at Target Genes for MSI
Twelve genes containing repeat sequences were chosen for study either because they are frequent targets for MSI-driven mutations in MSI-H tumors or because they play key roles in the maintenance of the genome integrity (BAX, BLM, GRB14, GRK4, IGF2R, MSH3, MSH6, RAD50, RECQL, TCF-4, TFDP2, and TGFBRII). PCR was performed with primers specific for each selected target gene (sequences are available on request).³⁵ Results obtained were compared with those already reported by our group in a larger series of MSI-H sporadic CRCs.³⁶

Statistical Analysis
Descriptive statistical analysis used ² tests and t tests. Two-by-two table contingency analyses were performed using a two-tailed Fisher's exact test because some numerical values were less than five. A P value of .05 was considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS Appendix REFERENCES

 
IBD Cohort
From 128 UC patients we collected 89 CRCs, 91 colorectal dysplastic lesions, and six colorectal adenomas; from 73 CD patients we collected 50 CRCs, 15 small bowel adenocarcinomas, 14 colorectal dysplastic lesions, and four colorectal and two small bowel adenomas. Four patients with indeterminate colitis suffering from CRC (n = 2) or dysplastic lesions of the colorectum (n = 2) were included in this series (Table 1). Information about the family history of cancer was available in 139 of the 205 patients. None had a family history of colorectal, gastric, or endometrial cancer suggestive of HNPCC.

Clinicopathologic Features of IBD-Ns According to MSI Status Compared With MSI-H Non-IBD CRCs
According to the presence or absence of MLH1 promoter methylation, the non-IBD colorectal MSI tumors were considered as likely sporadic (n = 18) or likely HNPCC (n = 15), respectively. MSI-H IBD-Ns showed clinical features more similar to those of likely HNPCC MSI-H CRCs as compared with likely sporadic MSI-H CRCs for age at diagnosis (52.4 years [range, 19 to 74 years] v 73.3 years [range, 48 to 92 years]; P = .0002), sex, and tumor localization (Table 2).

Data relating to IBD immunosuppressive medical treatment were available for 82 patients. When considering overall immunosuppressive regimens, there was no significant relationship between the occurrence of MSI and the intake of these drugs. Regarding azathioprine alone, there was no significant excess of neoplasia complicating IBD with MSI-H status as compared with MSS neoplasia in the group of patients treated with this drug (four of 10 patients with an MSI-H phenotype v 23 of 82 patients with an MSS phenotype; not significant). A similar percentage of patients received 5-aminosalicylic acid therapy in MSI-H IBD-Ns compared with MSS IBD-N (seven of nine patients with an MSI-H phenotype v 48 of 69 patients with an MSS phenotype; not significant).

Heterogeneous Primary MMR Defects in MSI-H IBD-Ns
Among the 24 tumors showing MMR defects by IHC (20 CRCs and four dysplastic lesions), MSI-H was found to be related to MLH1 deficiency in six CRCs and one dysplastic lesion (seven patients), was related to MSH2 deficiency in six CRCs and two dysplastic lesions (five patients), was related to MSH6 deficiency in six CRCs and one dysplastic lesion (two patients), and was related to PMS2 deficiency in two CRCs (two patients; Table 3). Thus, MMR defects observed in IBD-N MSI-H tumors are more closely related to what is generally observed in HNPCC MSI-H tumors than in sporadic MSI-H tumors (Fig 1A to 1D).

View larger version (148K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 1. Mismatch repair protein immunostaining in a mucinous colorectal cancer associated with Crohn's disease. This colorectal cancer is referred to as MLH1 deficient. (A) There is a loss of expression of MLH1 in neoplastic cells. (B) This tumor also shows loss of PMS2 protein expression; however, (C) MSH2 and (D) MSH6 are normally expressed.

MLH1 Promoter Methylation in MSI-H IBD-Ns
Among six MSI-H tumors showing MLH1 deficiency by IHC (five CRCs and one dysplastic lesion), methylation of the promoter of MLH1 was identified in three patients (three CRCs; Fig 2). This was associated with BRAF mutation in one patient (Table 3).

View larger version (13K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 2. MLH1 promoter methylation in microsatellite instability inflammatory bowel disease–associated intestinal neoplasias showing loss of MLH1 (samples 33, 3, 204, 188, 72, and 510) immunostaining. Neoplasias with loss of PMS2 immunostaining (samples 49 and 17) served as negative controls. M, methylated MLH1; U, unmethylated MLH1; Cm, methylated control; Cu, unmethylated control; water, water control lacking genomic DNA. (*) Samples with MLH1 promoter methylation.

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS Appendix REFERENCES

Our data reflect fundamental differences in primary MMR defects associated with MSI-H IBD-Ns as compared with sporadic MSI-H CRCs, showing heterogeneous MMR defects that involve MLH1, MSH2, MSH6, or PMS2. Notably, loss of MLH1 expression was found in only a minority of MSI-H IBD-Ns (six of 15; 40%). Moreover, whereas loss of MLH1 as a consequence of the methylation of its promoter site was reported to be the only mechanism leading to MMR deficiency in MSI-H IBD-Ns to date,^21-23 we report here that it occurs in only half of the MLH1-deficient MSI-H patient samples. The explanation for such a discrepancy is probably related to the fact that previous authors used nonspecific primers for the determination of MLH1 methylation status,³⁸ in contrast to this study.³⁴ Indeed, only a small proximal region of the MLH1 promoter (C region) has been demonstrated to harbor a methylation status that is correlated invariably with the loss of MLH1 expression.³⁹ In contrast, methylation outside this region does not always correlate with the lack of MLH1 expression. Data from our group corroborate these findings.²⁷ Interestingly, a recent study showed that DNA methylation, including MLH1 promoter methylation, was uncommon in UC cancers.⁴⁰

Conversely, MSI-H IBD-Ns present with a similar repertoire of secondary mutational events compared with MSI-H sporadic CRCs.³⁶ These include comparable frequencies of frameshift alterations affecting TGFβ-RII and other target genes for instability, as well as frequent BRAF-V600E activating mutations.¹⁰ Our results contradict those recently reported by Schulmann et al,²³ which showed a significantly lower frequency of TGFβ-RII frameshift and other (ACVR2) gene mutations in MSI-H IBD-associated CRCs compared with MSI-H sporadic CRCs. Additional studies of numerous target genes for instability in larger series of MSI-H IBD-Ns are required to determine if the repertoire of target genes involved in MSI-H IBD-Ns is genuinely different from that of sporadic MSI-H CRCs.

We also demonstrate here that MSI-H IBD-Ns are characterized by different clinical features compared with MSI-H sporadic CRCs, whereas some of these features (eg, age at diagnosis, sex, or tumor localization) resemble those seen with MSI-H cancers that occur in the HNPCC syndrome. Although the possibility of a familial association between IBD and HNPCC is not clearly defined,^41,42 it is highly unlikely that all patients with a MSI-H IBD-N included in this series would be HNPCC carriers. CRCs in patients with IBD occur at a young age,⁴³ and five patients met one of the extended Bethesda guidelines (CRC diagnosed at younger than 50 years). These findings highlight the fact that IBD could be a confounding factor for the diagnosis of HNPCC in some patients, with clinical implications for the genetic counseling of these patients. Thus, in light of the present data, we propose modifying the first and third Bethesda guidelines as follows: "1. CRC diagnosed in a patient <50 y of age in the absence of personal history of IBD" and "3. CRC with MSI-H phenotype diagnosed in a patient <60 y of age in the absence of personal history of IBD." In addition, our data highlight the necessity to perform MSI testing of IBD-Ns by studying the expression of MLH1, MSH2, MSH6, and PMS2 by IHC, whereas the study of MLH1 promoter methylation status alone would not be pertinent in that context. This last point is important because methylation is increasingly being proposed to distinguish MSI-H sporadic CRCs from HNPCC-related CRCs.

Chronic inflammation and immunosuppression have been proposed recently to constitute risk factors for the development of MSI-H cancers. We indeed found that both of these parameters were strongly associated with the occurrence of MSI in immunodeficiency-related non-Hodgkin's lymphomas.¹⁵ In another study, the intake of azathioprine, an immunosuppressive methylating agent, was proposed to increase the risk of developing acute myeloid leukemia/myelodysplastic syndrome exhibiting MSI,⁴⁴ through a phenomenon known as methylation tolerance.⁴⁵ Chronic inflammation induces oxidative stress, a state in which reactive oxygen species are produced in increased abundance and modify DNA structures.⁴⁶ Recently, an adaptive and imbalanced increase in the activities of base-excision repair proteins was shown to generate MSI in inflamed, non-neoplastic UC specimens.⁴⁷ Taking account of these observations, MSI in our study was not found to be associated with the intake of immunosuppressive regimens and occurred in patients who had never been treated with azathioprine. Accordingly, we suggest that MSI would mostly be favored in IBD through chronic inflammation leading to inactivation of the MMR system by mechanisms that do not relate to epigenetic and age-related silencing of the MLH1 gene. Additional studies are necessary to determine the molecular pathways leading to MMR deficiency in that context. Of interest, heterogeneous MMR defects with a low incidence of MLH1 methylation also characterize MSI-H post-transplantation non-Hodgkin's lymphomas (Borie et al, manuscript in preparation), suggesting that such pathways would be related to both of these new subtypes of MSI-H neoplasms and probably others whose incidence is favored in related clinical contexts. This makes it important to perform MSI testing in large series of other types of inflammation- or immunosuppression-related neoplasms.

	AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS Appendix REFERENCES

 
The author(s) indicated no potential conflicts of interest.

	AUTHOR CONTRIBUTIONS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS Appendix REFERENCES

 
Conception and design: Richard Hamelin, Alex Duval, Jean-François Fléjou

Provision of study materials or patients: Céline Bossard, Françoise Berger, Emmanuelle Leteurtre, Anne Lavergne-Slove, Marie-Pierre Chenard, Emmanuel Tiret

Collection and assembly of data: Magali Svrcek, Alexandra Chalastanis, Emilie Capel, Sylvie Dumont, Olivier Buhard, Carla Oliveira, Raquel Seruca, Jean-François Mosnier, Jacques Cosnes, Laurent Beaugerie

Data analysis and interpretation: Magali Svrcek, Jamila El-Bchiri, Emilie Capel, Olivier Buhard, Carla Oliveira, Raquel Seruca, Richard Hamelin, Alex Duval, Jean-François Fléjou

Manuscript writing: Magali Svrcek, Alex Duval, Jean-François Fléjou

Final approval of manuscript: Magali Svrcek, Jamila El-Bchiri, Alexandra Chalastanis, Emilie Capel, Sylvie Dumont, Olivier Buhard, Carla Oliveira, Raquel Seruca, Céline Bossard, Jean-François Mosnier, Françoise Berger, Emmanuelle Leteurtre, Anne Lavergne-Slove, Marie-Pierre Chenard, Richard Hamelin, Jacques Cosnes, Laurent Beaugerie, Emmanuel Tiret, Alex Duval, Jean-François Fléjou

	Appendix

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS Appendix REFERENCES

 

	ACKNOWLEDGMENTS

 
We thank Dr Barry Iacopetta for critical reading of the manuscript. We also thank Dr Bordahandy, cabinet d'Anatomie Pathologique, Biarritz, France; center hospitalier de Niort, avenue Charles de Gaulle, Niort, France; Professor Patrice Callard, Assistance Publique-Hôpitaux de Paris (AP-HP), Service d'Anatomie et Cytologie Pathologiques, Hôpital Tenon, Paris, France; Dr Denis Chatelain, Service d'Anatomie et Cytologie Pathologiques, Hôpital Nord, Amiens, France; Dr Anne Couvelard, AP-HP, Service d'Anatomie et Cytologie Pathologiques, Hôpital Beaujon, Clichy, France; Professor Marie-Danielle Diébold, Laboratoire central d'Anatomie et Cytologie Pathologiques, Hôpital Robert Debré, Reims, France; Professor Bernard Flourié, Service d'Hépato-Gastroentérologie, Centre Hospitalier Lyon Sud, France; Dr Yves François, Service de Chirurgie Générale et Digestive; Centre Hospitalier Lyon Sud, France; Dr Isabelle Kleinclaus, Service d'Histologie et Cytologie Pathologiques, Hôpital Pasteur, Colmar, France; Dr Thierry Lazure, AP-HP, Service central d'Anatomie et Cytologie Pathologiques, Le Kremlin-Bicêtre, France; Dr Philippe Rouvier, Service d'Anatomie et Cytologie Pathologiques, Centre hospitalier intercommunal André Grégoire, Montreuil, France; Professor Jean-Yves Scoazec, Hospices Civils de Lyon, Hôpital Edouard Herriot, France; Dr Anne-Sophie Thirouard, Service d'Anatomie et Cytologie Pathologiques, Centre Hospitalier et Universitaire de Rennes, Pontchaillou, Rennes, France; Dr Pierre Validire, Département d'Anatomie Pathologique, Institut Mutualiste Montsouris, Paris, France, for providing samples.

	NOTES

 
Supported in part by grants from the Association Nationale de Recherche sur le SIDA (Credit No. 03/162) and from the Association pour la Recherche contre le Cancer (Credit No. 3301). J.E.B. and A.C. were recipients of a fellowship from the Ministère Français de la Recherche.

Authors’ disclosures of potential conflicts of interest and author contributions are found at the end of this article.

	REFERENCES

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS Appendix REFERENCES

 
1. Ionov Y, Peinado MA, Malkhosyan S, et al: Ubiquitous somatic mutations in simple repeated sequences reveal a new mechanism for colonic carcinogenesis. Nature 363:558-561, 1993[CrossRef][Medline]

2. Thibodeau SN, Bren G, Schaid D: Microsatellite instability in cancer of the proximal colon. Science 260:816-819, 1993[Abstract/Free Full Text]

3. Aaltonen LA, Peltomaki P, Leach FS, et al: Clues to the pathogenesis of familial colorectal cancer. Science 260:812-816, 1993[Abstract/Free Full Text]

4. Jass JR, Do K-A, Simms LA, et al: Morphology of sporadic colorectal cancer with DNA replication errors. Gut 42:673-679, 1998[Abstract/Free Full Text]

5. Boland CR, Thibodeau SN, Hamilton SR, 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 58:5248-5257, 1998[Abstract/Free Full Text]

6. Hampel H, Frankel WL, Martin E, et al: Screening for the Lynch Syndrome (Hereditary Non-Polyposis Colorectal Cancer). N Engl J Med 352:1851-1860, 2005[Abstract/Free Full Text]

7. Veigl ML, Kasturi L, Olechnowicz J, et al: Biallelic inactivation of hMLH1 by epigenetic gene silencing, a novel mechanism causing human MSI cancers. Proc Natl Acad Sci U S A 95:8698-8702, 1998[Abstract/Free Full Text]

8. Davies H, Bignell GR, Cox C, et al: Mutations of the BRAF gene in human cancers. Nature 417:949-954, 2002[CrossRef][Medline]

9. Rajagopalan H, Bardelli A, Lengauer C, et al: Tumorigenesis: RAF/RAS oncogenes and mismatch-repair status. Nature 418:934, 2002[CrossRef][Medline]

10. Oliveira C, Pinto M, Duval A, et al: BRAF mutations characterize colon but not gastric cancer with mismatch repair deficiency. Oncogene 22:9192-9196, 2003[CrossRef][Medline]

11. Wang L, Cunningham JM, Winters JL, et al: BRAF mutations in colon cancer are not likely attributable to defective DNA mismatch repair. Cancer Res 63:5209-5212, 2003[Abstract/Free Full Text]

12. Domingo E, Laiho P, Ollikainen M, et al: BRAF screening as a low-cost effective strategy for simplifying HNPCC genetic testing. J Med Genet 41:664-668, 2004[Abstract/Free Full Text]

13. Domingo E, Niessen RC, Oliveira C, et al: BRAF-V600E is not involved in the colorectal tumorigenesis of HNPCC in patients with functional MLH1 and MSH2 genes. Oncogene 24:3995-3998, 2005[CrossRef][Medline]

14. 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 62:2447-2454, 2002[Abstract/Free Full Text]

15. Duval A, Raphael M, Brennetot C, et al: The mutator pathway is a feature of immunodeficiency-related lymphomas. Proc Natl Acad Sci U S A 101:5002-5007, 2004[Abstract/Free Full Text]

16. Suzuki H, Harpaz N, Tarmin L, et al: Microsatellite instability in ulcerative colitis-associated colorectal dysplasias and cancers. Cancer Res 54:4841-4844, 1994[Abstract/Free Full Text]

17. Brentnall TA, Crispin DA, Bronner MP, et al: Microsatellite instability in nonneoplastic mucosa from patients with chronic ulcerative colitis. Cancer Res 56:1237-1240, 1996[Abstract/Free Full Text]

18. Willenbucher RF, Aust DE, Chang CG, et al: Genomic instability is an early event during the progression pathway of ulcerative-colitis-related neoplasia. Am J Pathol 154:1825-1830, 1999[Abstract/Free Full Text]

19. Lyda MH, Noffsinger A, Belli J, et al: Microsatellite instability and K-ras mutations in patients with ulcerative colitis. Hum Pathol 31:665-671, 2000[CrossRef][Medline]

20. Cawkwell L, Sutherland F, Murgatroyd H, et al: Defective hMSH2/hMLH1 protein expression is seen infrequently in ulcerative colitis associated colorectal cancers. Gut 46:367-369, 2000[Abstract/Free Full Text]

21. Fleisher AS, Esteller M, Harpaz N, et al: Microsatellite instability in inflammatory bowel disease-associated neoplastic lesions is associated with hypermethylation and diminished expression of the DNA mismatch repair gene, hMLH1. Cancer Res 60:4864-4868, 2000[Abstract/Free Full Text]

22. Aust DE, Haase M, Dobryden L, et al: Mutations of the BRAF gene in ulcerative colitis-related colorectal carcinoma. Int J Cancer 115:673-677, 2005[CrossRef][Medline]

23. Schulmann K, Mori Y, Croog V, et al: Molecular phenotype of inflammatory bowel disease-associated neoplasms with microsatellite instability. Gastroenterology 129:74-85, 2005[CrossRef][Medline]

24. Umetani N, Sasaki S, Watanabe T, et al: Genetic alterations in ulcerative colitis-associated neoplasia focusing on APC, K-ras gene and microsatellite instability. Jpn J Cancer Res 90:1081-1087, 1999[CrossRef]

25. Holzmann K, Klump B, Borchard F, et al: Comparative analysis of histology, DNA content, p53 and Ki-ras mutations in colectomy specimens with long-standing ulcerative colitis. Int J Cancer 76:1-6, 1998[CrossRef][Medline]

26. Vasen HFA, Watson P, Mecklin J-P, et al: New clinical criteria for hereditary nonpolyposis colorectal cancer (HNPCC, Lynch syndrome) proposed by the International Collaborative Group on HNPCC. Gastroenterology 116:1453-1456, 1999[CrossRef][Medline]

27. Capel E, Fléjou J-F, Hamelin R: Assessment of MLH1 promoter methylation in relation to gene expression requires specific analysis. Oncogene [epub ahead of print on June 4, 2007]

28. Goelz SE, Hamilton SR, Vogelstein B: Purification of DNA from formaldehyde fixed and paraffin embedded human tissue. Biochem Biophys Res Commun 130:118-126, 1985[CrossRef][Medline]

29. Buhard O, Suraweera N, Lectard A, et al: Quasimonomorphic mononucleotide repeats for high level microsatellite instability analysis. Dis Markers 20:251-257, 2004[Medline]

30. Rigau V, Sebbagh N, Olschwang S, et al: Microsatellite instability in colorectal carcinoma: The comparison of immunohistochemistry and molecular biology suggests a role for hMSH6 immunostaining. Arch Pathol Lab Med 127:694-700, 2003[Medline]

31. Hamilton SR, Vogelstein B, Kudo S, et al: Carcinoma of the colon and rectum. In: Hamilton SR, Aaltonen LA, eds. Pathology and Genetics: Tumours of the digestive system—World Health Organization Classification of Tumuors. Lyon, France, IARC Press, 2000, pp 105-119

32. Schlemper RJ, Riddell RH, Kato Y, et al: The Vienna classification of gastrointestinal epithelial neoplasia. Gut 47:251-255, 2000[Abstract/Free Full Text]

33. Frommer M, McDonald LE, Millar DS, et al: Genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc Natl Acad Sci U S A 89:1827-1831, 1992[Abstract/Free Full Text]

34. Park SJ, Rashid A, Lee JH, et al: Frequent CpG island methylation in serrated adenomas of the colorectum. Am J Pathol 162:815-822, 2003[Abstract/Free Full Text]

35. Jacob S, Miquel C, Sarasin A, et al: Effects of camptothecin on double-strand break repair by non-homologous end-joining in DNA mismatch repair-deficient human colorectal cancer cell lines. Nucleic Acid Res 33:106-113, 2005[Abstract/Free Full Text]

36. Duval A, Rolland S, Compoint A, et al: Evolution of instability at coding and non-coding repeat sequences in human MSI-H colorectal cancers. Hum Mol Genet 10:513-518, 2001[Abstract/Free Full Text]

37. Suraweera N, Duval A, Reperant M, et al: Evaluation of tumor microsatellite instability using 5 quasimonomorphic mononucleotide repeats and pentaplex PCR. Gastroenterology 123:1804-1811, 2002[CrossRef][Medline]

38. Herman JG, Umar A, Polyak K, et al: Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma. Proc Natl Acad Sci U S A 95:6870-6875, 1998[Abstract/Free Full Text]

39. Deng G, Chen A, Hong J, et al: Methylation of CpG in a small region of the hMLH1 promoter invariably correlates with the absence of gene expression. Cancer Res 59:2029-2033, 1999[Abstract/Free Full Text]

40. Konishi K, Shen L, Wang S, et al: Rare CpG island methylator phenotype in ulcerative colitis-associated neoplasias. Gastroenterology 132:1254-1260, 2007[CrossRef][Medline]

41. Caruso ML, Cristofaro G, Lynch HT: HNPCC-Lynch syndrome and idiopathic inflammatory bowel disease: A hypothesis on sharing of genes. Anticancer Res 17:2647-2649, 1997[Medline]

42. Sandborn WJ: Inflammatory bowel disease and hereditary nonpolyposis colorectal cancer: Is there a genetic link? Gastroenterology 114:608-609, 1998[CrossRef][Medline]

43. Bernstein CN, Blanchard JF, Kliewer E, Wajda A: A cancer risk in patients with inflammatory bowel disease: A population-based study. Cancer 91:854-862, 2001[CrossRef][Medline]

44. Offman J, Opelz G, Doehler B, et al: Defective DNA mismatch repair in acute myeloid leukemia/myelodysplastic syndrome after organ transplantation. Blood 104:822-828, 2004[Abstract/Free Full Text]

45. Branch P, Aquilina G, Bignami M, et al: Defective mismatch binding and a mutator phenotype in cells tolerant to DNA damage. Nature 362:652-654, 1993[CrossRef][Medline]

46. Marnett LJ: Oxyradicals and DNA damage. Carcinogenesis 21:361-370, 2000[Abstract/Free Full Text]

47. Hofseth LJ, Khan MA, Ambrose M, et al: The adaptative imbalance in base excision-repair enzymes generates microsatellite instability in chronic inflammation. J Clin Invest 112:1887-1894, 2003[CrossRef][Medline]

Submitted January 29, 2007; accepted June 20, 2007.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Facebook    Reddit    Technorati    Twitter    What's this?

This article has been cited by other articles:

				S. Ogino, K. Nosho, G. J. Kirkner, K. Shima, N. Irahara, S. Kure, A. T. Chan, J. A. Engelman, P. Kraft, L. C. Cantley, et al. PIK3CA Mutation Is Associated With Poor Prognosis Among Patients With Curatively Resected Colon Cancer J. Clin. Oncol., March 20, 2009; 27(9): 1477 - 1484. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Save to my personal folders Download to citation manager Rights & Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Svrcek, M. Articles by Fléjou, J.-F. Search for Related Content PubMed PubMed Citation Articles by Svrcek, M. Articles by Fléjou, J.-F. Social Bookmarking                            What's this?