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Clinical Cancer Research 13, 2865-2869, May 15, 2007. doi: 10.1158/1078-0432.CCR-06-2174
© 2007 American Association for Cancer Research
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Human Cancer Biology

Microsatellite Instability Markers for Identifying Early-Onset Colorectal Cancers Caused by Germ-Line Mutations in DNA Mismatch Repair Genes

Leeanne J. Mead1, Mark A. Jenkins2, Joanne Young5, Simon G. Royce1, Letitia Smith1, D. James B. St. John3, Finlay Macrae3, Graham G. Giles4, John L. Hopper2 and Melissa C. Southey1,6

Authors' Affiliations: 1 Genetic Epidemiology Laboratory, Department of Pathology and 2 Centre for Molecular, Environmental, Genetic and Analytic Epidemiology, The University of Melbourne; 3 Department of Colorectal Medicine and Genetics, The Royal Melbourne Hospital; 4 Centre for Cancer Epidemiology, The Cancer Council Victoria, Melbourne, Victoria, Australia; 5 Molecular Cancer Epidemiology, Queensland Institute of Medical Research, Brisbane, Queensland, Australia; and 6 The IARC, Lyon, France

Requests for reprints: John L. Hopper, Centre for Molecular, Environmental, Genetic and Analytic Epidemiology, The University of Melbourne, Melbourne, Victoria, 3010 Australia. Phone: 61-3-8344-0697; Fax: 61-3-9347-9824; E-mail: j.hopper{at}unimelb.edu.au


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Microsatellite instability (MSI) testing of colorectal cancer tumors is used as a screening tool to identify patients most likely to be mismatch repair (MMR) gene mutation carriers. We wanted to examine which microsatellite markers currently used to detect MSI best predict early-onset colorectal cancer caused by germ-line mutations in MMR genes.

Experimental Design: Invasive primary tumors from a population-based sample of 107 cases of colorectal cancer diagnosed before age 45 years and tested for germ-line mutations in MLH1, MSH2, MSH6, and PMS2 and MMR protein expression were screened for MSI using the National Cancer Institute panel and an expanded 10-microsatellite marker panel.

Results: The National Cancer Institute five-marker panel system scored 31 (29%) as NCIMSI-High, 13 (12%) as NCIMSI-Low, and 63 (59%) as NCIMS-Stable. The 10-marker panel classified 18 (17%) as 10MSI-High, 17 (16%) as 10MSI-Low, and 72 (67%) as 10MS-Stable. Of the 26 cancers that lacked the expression of at least one MMR gene, 24 (92%) were positive for some level of MSI (using either microsatellite panel). The mononucleotide repeats Bat26, Bat40, and Myb were unstable in all 10MSI-High cancers and all MLH1 and MSH2 mutation carriers (100% sensitive). Bat40 and Bat25 were unstable in all tumors of MSH6 mutation carriers (100% sensitive). Bat40 was unstable in all MMR gene mutation carriers (100% sensitive). By incorporating seven mononucleotide repeats markers into the 10-marker panel, we were able to distinguish the carriers of MSH6 mutations (all scored 10MSI-Low) from the MLH1 and MSH2 mutation carriers (all scored 10MSI-High).

Conclusions: In early-onset colorectal cancer, a microsatellite panel containing a high proportion of mononuclear repeats can distinguish between tumors caused by MLH1 and MSH2 mutations from those caused by MSH6 mutations.

Only a small proportion of early-onset colorectal cancer is thought to be due to mutations in mismatch repair (MMR) genes, perhaps <20% (1). Not all individuals with early-onset colorectal cancer (i.e., diagnosed under the age of 45 years) who carry mutations in these genes have a family history sufficient for classification as hereditary nonpolyposis colorectal cancer (1, 2), and this observation is rarely due to de novo mutations (3, 4). However, once identified, MMR gene mutation carriers and their families can benefit from improved clinical management that includes appropriate surveillance procedures, treatment options, and predictive testing for unaffected family members.

Given that genetic testing for germ-line mutations in MMR genes is technically challenging and costly, screening strategies have been developed to better identify the individuals with colorectal cancer who are most likely to carry a germ-line MMR gene mutation. These strategies include examining the expression of MMR proteins in tumor material using immunohistochemistry, testing for tumor microsatellite instability (MSI), and taking a family history of colorectal cancer and other cancers thought to be caused by MMR mutations (1, 5, 6). However, practical and local issues, such as the availability of sufficient laboratory-based expertise, may limit the application of these strategies. Therefore, refining and understanding the potential of each strategy as a stand-alone screening method for identifying likely carriers of MMR gene mutations requires further attention. In this study, we have focused on MSI testing.

Although the definition of MSI and methodologies applied to detect it, including the nature and number of microsatellite markers used, have varied across studies (1, 7–9), it is thought that ~15% of all colorectal cancer (10) and ~30% of early-onset colorectal cancer (1) display some degree of MSI. However, only a small proportion (10-20%) of all colorectal cancer that display MSI have identifiable underlying mutations in MMR genes. A substantial proportion of MSI-positive tumors are due to MLH1 promoter methylation, particularly the late-onset cases (11, 12).

In a recent study of early-onset colorectal cancer, we reported that having a MSI-High tumor had a positive predictive value of 0.7 (95% confidence interval, 0.5-0.9) for having an identifiable germ-line mutation in any of four MMR genes: MLH1, MSH2, MSH6, or PMS2 (1). In a study of MSI in a hospital series of colorectal cancer, examination of just mononucleotide repeat markers was sufficient for the identification of tumors with MMR defects, as defined by lack of immunohistochemical staining for MLH1, MSH2, MSH6, and PMS2 (8). However, that study did not report germ-line MMR gene status of these cancers. As the identification of individuals with germ-line MMR mutations is the important clinical end point (rather than simply the identification of MMR deficiency), the findings of this report may be difficult to interpret.

Unfortunately, there has been great variability in markers that have been used in reports of MSI in colorectal cancer and, as there is evidence that the sensitivity and specificity of markers to detect MSI differ across markers, it is not easy to compare and combine published data. The precise composition of an ideal panel for the identification of MSI in MMR gene mutation carriers continues to evolve. Some reports have suggested that the poly-A repeat in Bat26 is alone sufficient for MSI testing (13, 14) but others have shown variability (see refs. 8, 15 for examples). There is also evidence from the MSI testing of extracolonic hereditary nonpolyposis colorectal cancer–related tumors that microsatellites display tissue-specific vulnerability to instability in the presence of MMR gene mutations (16, 17). A five-microsatellite marker panel for the detection of MSI was recommended in a report of a meeting on MSI at the National Cancer Institute (NCI) in 1998 (7). This panel, the "NCI panel," has been used extensively in clinical and research settings.

Here, we report the details of MSI testing done using a 10-microsatellite marker panel in a population-based, case-family study of early-onset colorectal cancer conducted in Victoria (Australia). We compare our 10-marker panel with the NCI panel for specificity and sensitivity of MMR protein expression and germ-line MMR gene mutation status.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Subjects. The Victorian Colorectal Cancer Family Study is a population-based, case-control family study of early-onset colorectal cancer, conducted from 1993 to 1997 on 131 cases of histologically verified primary adenocarcinomas of the colon or rectum registered on the Victorian Cancer Registry (1, 18). The probands (all diagnosed between 18 and 44 years of age) consisted of 66 females and 65 males, with the mean age at diagnosis being 39.5 years (SD, 4.7 years). Probands and their living relatives were asked to provide a blood sample and sign an informed consent, to allow access to their tumor tissues from pathology laboratories for use in molecular analyses. The study was approved by the ethics committees of The University of Melbourne and The Cancer Council Victoria.

Tumor specimens. Samples of invasive tumors from primary colorectal adenocarcinoma, and from other cancer diagnoses when available, were obtained from hospitals and private pathology laboratories for 118 (90%) case patients. Six probands did not consent to release tissue to the study and two laboratories had not agreed to release the remaining seven samples at the time of this study. All tumor samples were reviewed by a pathologist who directed the microdissection.

Testing for tumor MSI. MSI testing was done on invasive tumor cells microdissected from 5-µm sections of paraffin-embedded archival tumor tissue stained with 1% methyl green. DNA extracted from histologically normal cells microdissected from colonic or lymph node tissue, or DNA extracted from peripheral blood lymphocytes, was used to control the assay as described previously (1, 16, 19). Ten microsatellite markers were assessed: 3 dinucleotide repeats (D5S346, D17S250, and D2S123) and 7 mononucleotide repeats (BAT25, BAT26, BAT40, MYB, TGFßRII, IGFIIR, and BAX). For the purpose of this study, the degree of instability in each tumor was scored in two ways. The first used data from all 10 markers to categorize tumors as microsatellite stable (10MS-Stable), low (10MSI-Low), and high (10MSI-High) when 0 to 1, 2 to 5, and 6 to 10 markers, respectively, were identified as unstable (1, 16). The second, as recommended by the NCI Workshop on MSI, used a five-marker panel to classify tumors as NCIMSI-High, NCIMSI-Low, or NCIMS-Stable defining tumors as MSI-High if two or more of the five markers show instability (i.e., have insertion/deletion mutations) and MSI-Low if only one of the five markers shows instability (7). Assessment of MSI was not successful for 11 (10%) tumor samples due to technical reasons related to tumor DNA quality, leaving 107 tested tumors, which included 106 colorectal, and one stomach tumor (collected from a case where the colorectal cancer could not be located at the diagnostic laboratory).

Testing for germ-line MMR gene mutations. As reported previously in detail (1), all exonic and flanking intronic sequences of the MLH1, MSH2, MSH6, and PMS2 were screened for germ-line mutations using sequencing approaches, except for exon 4 of MSH6 that was screened in eight overlapping fragments using denaturing high-pressure liquid chromatography. The multiplex ligation-dependent probe amplification assay (MCR-Holland; ref. 20) to detect large genomic alterations in MLH1 and MSH2 was performed on samples from probands that had tumors lacking at least one MMR protein expression and for which no previous mutation had been identified by sequencing. Break points were characterized by long-range PCR encompassing the predicted genomic alteration, cloning, and sequencing (3). Mutation testing was conducted for all case patients with the one or more of the following characteristics: (a) having a family history that fulfilled the Amsterdam Criteria for hereditary nonpolyposis colorectal cancer, (b) having a tumor that was MSI-High, or MSI-Low, or that lacked expression of at least one MMR protein; and (c) being in a random sample of 23 selected from those who had tumors that were MS-Stable and did not lack expression of any MMR protein. Of the 18 mutations detected, 15 (83%) were predicted to produce a truncated protein product (4 were alterations in splice site regions, 7 were frameshift mutations, 2 were nonsense mutations, and 2 were large deletions). The remaining three were a missense mutation, and two small in-frame deletions all considered deleterious (1).

Testing for tumor expression of MMR genes. The expression of MLH1, MSH2, MSH6, and PMS2 was assessed by immunohistochemistry testing on 3-µm formalin-fixed paraffin-embedded sections using standard methods, monoclonal antibodies MLH1 (PharMingen clone G168-728), MSH2 (Oncogene clone FE11), MSH6 (BD Transduction Laboratories clone 44), and PMS2 (PharMingen clone A16-4) on a DAKO autostainer. Normal colonic epithelium adjacent to tumor and lymphocytes served as inbuilt positive controls. A gastrointestinal pathologist scored the tumors as positive when nuclear staining in tumor tissue was present, or otherwise as negative.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Table 1 shows the results of the MSI testing using both the 10-marker and the NCI (5 markers) panel scoring systems. Of the 17 tumors scored as 10MSI-Low, only 3 were scored as NCIMSI-Low (cases 74-76) and the others were NCIMSS (n = 1, case 73) and NCIMSI-High (n = 13, cases 77-89). Of the 13 tumors scored as NCIMSI-Low, 3 were also scored as 10MSI-Low (cases 74-76) and the remaining 10 scored as 10MSS (cases 63-72; Fig. 1 ; Table 1).


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  		Table 1. Characteristics of MSI-positive tumors, including MMR protein expression and germ-line MMR gene status

 

Figure 1
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  		Fig. 1. MSI scores using the 10-microsatellite marker panel and the NCI panel. A, the MSI results using the 10-microsatellite marker panel. B, the MSI results using the NCI 5-marker panel. The number of tumors in each MSI category, the proportion of tumors from MMR gene mutation carriers, designation of MS-Stable (S), MSI-Low (L), and MSI-High (H) is indicated for each of the 107 early-onset colorectal cancers.

 
Table 1 shows that 24 of the 26 (92%) cancers that lacked the expression of at least one of the MMR genes were positive for MSI. All four tumors arising in individuals with germ-line mutations in MSH6 lacked the protein expression of MSH6 and were scored as 10MSI-Low and NCIMSI-High. None of these MSH6 mutation carriers had more than one unstable dinucleotide repeat.

Forty-nine (46%) of the primary early-onset cancers screened for MSI had one or more unstable markers. Table 2 shows that the most frequently unstable marker was Bat40, found to be unstable in 28 (26%) tumor DNA samples. The microsatellite marker found to be least frequently unstable was IGFIIR (6 of 107, 6%). Bat26, Bat40, and Myb were found to be unstable in all the 10MSI-High cancers and thus in all the MLH1 and MSH2 mutation carriers (100% sensitive). Bat40 was also unstable in the tumors in cases known to carry MSH6 mutations and thus was unstable in all 17 known MMR gene mutation carriers (100% sensitive).


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  		Table 2. Frequency of instability observed in each microsatellite marker

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
How specific and sensitive can MSI testing of colorectal cancer diagnosed at an early age (before 45 years) be for the identification of MMR gene mutations carriers? We have examined this question using a population-based case-family study of colorectal cancer diagnosed before the age of 45 years and found that MSI had a predictive value of 0.7 (95% confidence interval, 0.5-0.9) for having an identifiable germ-line mutation in any of four MMR genes (1).

Dissecting the MSI test into microsatellite marker components revealed some interesting aspects. Expansion of the microsatellite panel to include additional mononucleotide repeats was advantageous. Similarly to Hatch et al. (8), who reported that examination of mononucleotide repeats was sufficient for the identification of tumors with MMR defects, we found that the four microsatellite markers most sensitive to the detection of MSI in MMR gene mutation carriers were the mononucleotide repeats Bat40, Bat26, Bat25, and Myb; they detected MSI in 100%, 94%, 94%, and 88% of the carriers, respectively. The persistence of these mononucleotide markers to detect MSI in the group defined as 10MSI-Low (and the poor sensitivity of dinucleotide repeats to detect MSI in this group) enable the identification of a 10MSI-Low group that was predominantly mononucleotide unstable and contained all the MSH6 mutation carriers (cases 73-89; Table 1). As presented and discussed previously (1, 3, 21), the group of tumors defined as 10MSI-Low have a significant level of MSI that may signal an underlying molecular mechanism/pathway of tumorigenesis. This level of MSI is quite distinct from both the 10MSI-High, NCIMSI-High and the NCIMSI-Low classifications. In our study, mononucleotide markers were sufficient to detect all the tumors with MMR mutations; however, it was the failure of the dinucleotide repeat markers to display consistent MSI in the 10MSI-Low group that made them distinguishable; thus, there was great benefit to having them remain in the panel (Fig. 1; Tables 1 and 2).

This study specifically addresses MSI testing in colorectal cancer diagnosed before the age of 45 years. Our previous work characterizing this group of colorectal cancers has shown that several of the key molecular events of the carcinogenic pathways known to be associated with colorectal cancer are less frequently observed in early-onset disease. For example, lack of MHL1 expression in tumor samples (associated with MLH1 promoter methylation) is observed frequently (50%) in studies of all or later-onset colorectal cancer (11, 12, 22). We identified that only 3% of the early-onset cases lacked expression of MHL1 (via immunohistochemistry) and lacked the identification of a germ-line MMR gene mutation, suggesting promoter methylation (1). We have also found that the frequency of somatic K-ras mutations in early-onset colorectal cancer carcinogenesis is very low (21). The optimal MSI testing panel for later-onset colorectal cancer, which has a different molecular background to early-onset disease especially in the context of MLH1 promoter methylation, may not be the same as the optimal panel for early-onset colorectal and requires further investigation.


   Acknowledgments
 
We thank Judi Maskiell and the research interviewers; Gillian Dite and the data management staff; Sabar Napaki, Garry Grubb, Deon Venter, and Jane Armes for pathology input; and the men and women who participated in this study.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 8/31/06; revised 11/22/06; accepted 12/22/06.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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  17. Davalos V, Dopeso H, Velho S, et al. High EPHB2 mutation rate in gastric but not endometrial tumors with microsatellite instability. Oncogene 2007;26:308–11.[CrossRef][Medline]
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Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
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