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Evaluation of Microsatellite Instability, hMLH1 Expression and hMLH1 Promoter Hypermethylation in Defining the MSI Phenotype of Colorectal Cancer

Evaluation of Microsatellite Instability, hMLH1 Expression and hMLH1 Promoter Hypermethylation in... [Cancer Biology & Therapy 3:1, 73-78, January 2004]; ©2004 Landes Bioscience Research Paper Evaluation of Microsatellite Instability, hMLH1 Expression and hMLH1 Promoter Hypermethylation in Defining the MSI Phenotype of Colorectal Cancer 1,† ABSTRACT Christian N. Arnold 1,† Introduction: About 15% of all colorectal cancers (CRCs) demonstrate high levels of Ajay Goel microsatellite instability (MSI-H) and are currently best identified by molecular analysis of Carolyn Compton microsatellite markers. Most sporadic CRCs with MSI-H are known to be associated with the methylation of the hMLH1 promoter. Promoter methylation coincided with lack of Victoria Marcus hMLH1 expression. We aimed to investigate the association between MSI status, hMLH1 Donna Niedzwiecki protein expression and methylation status of the hMLH1 promoter, and to determine the use- fulness of each method in defining the MSI phenotype in sporadic CRCs. Materials and Jeannette M. Dowell Methods: CRCs from 173 patients from the Cancer and Leukemia Group B (CALGB) were Linda Wasserman assessed for their MSI status. An additional cohort of 18 MSI-H tumors from the University of California San Diego (UCSD) was included in the analysis of the MSI-H subgroup. Toru Inoue MSI testing was performed by PCR using five standard MSI markers. hMLH1 promoter Robert J. Mayer analysis was investigated by methylation specific PCR (MSP), and expression of the MMR 5 genes hMLH1 and hMSH2 was examined by immunohistochemistry (IHC). Results: Of the Monica M Bertagnolli 173 CALGB tumors, 111 (64%) were MSS, 35 (20%) were MSI-L and 27 (16%) MSI-H, 1,6, C. Richard Boland * respectively. Data on hMLH1 protein expression, hMSH2 protein expression and hMLH1 methylation are available on 128, 173 and 81 of these tumors, respectively. Presence of Department of Medicine and Cancer Center; University of California San Diego; La hMLH1 and hMSH2 protein expression was significantly associated with MSI status. Four Jolla, California USA of 45 (8.9%) MSI-H tumors and 0 of 146 (0%) MSS/MSI-L tumors did not express hMSH2 Department of Pathology; McGill University; Montreal, Quebec, Canada (p = 0.0028). hMLH1 protein expression was present in 107 of 108 (99%) MSS and CALGB Statistical Center; Duke University; Durham, North Carolina USA MSI-L tumors versus 11 of 20 (55%) MSI-H tumors (p < 0.0001). Of 61 MSS and MSI-L 4 cancers studied for methylation, 11 (18%) were methylated at the hMLH1 promoter Department of Medicine; Dana Farber Cancer Institute; Boston, Massachusetts USA whereas 14 of 20 (70%) MSI-H cancers were methylated (p = 0.0001). In 27 MSI-H Brigham and Women’s Hospital; Boston, Massachusetts USA tumors studied for hMLH1 protein expression and methylation, 93% of tumors with loss San Diego Veteran Affairs Medical Center; La Jolla, California USA of expression (93%) were also methylated while 42% (5/12) with positive immunostain- ing for hMLH1 were methylated at the hMLH1 promoter (p = 0.009). Conclusions: These authors contributed equally to this study Promoter methylation and hMLH1 expression are significantly associated with the MSI-H *Correspondence to: C. Richard Boland; Baylor University Medical Center; 2 phenotype in CRC. Promoter methylation analysis provides a useful means to screen for Hoblitzelle, 3500 Gaston Avenue; Dallas, Texas USA; Tel.: 214.820.2692; Fax: 214.818.9292; Email: RickBo@BaylorHealth.Edu MSI-H tumors. Our data further suggests that hMLH1 promoter methylation analysis alone cannot replace MSI testing, as a significant number of MSI-H tumors could be potentially Received 07/29/03; Accepted 09/05/03 overseen by such an approach. We suggest that phenotypic evaluation of CRC is performed Previously published online as a Cancer Biology & Therapy E-publication: most reliably with MSI testing, although expression analysis and investigation of the promoter http://www.landesbioscience.com/journals/cbt/abstract.php?id=590 methylation status may complement the screening process. KEY WORDS hMLH1, microsatellite instability, promoter methylation, colorectal cancer, immunohistochem- INTRODUCTION istry CRC pathogenesis proceeds through two well defined pathways of genomic instability, ACKNOWLEDGEMENTS termed as the suppressor and mutator pathways. These pathways are characterized by successive accumulation of genetic events in neoplastic cells as a function of time. In most The research for CALGB study 9865 was sup- human neoplasias, the first mechanism is observed at the chromosomal level and leads to ported, in part, by grants from the National Cancer Institute (CA31946) to the Cancer and Leukemia gene alterations by chromosomal gains and losses, accompanied by mutations at specific Group B (Richard L. Schilsky, Chairman). This tumor suppressor genes and oncogenes. The second mechanism was first described in work was also sponsored by a grant from the NIH 1993 by observing size variations of short repetitive DNA sequences in tumor DNA, (RO1-CA 72851) to C.R.B and in part by an 2-4 termed microsatellites. It was soon linked to hereditary non-polyposis colon cancer American Cancer Society-IRG award to A.G. (HNPCC) and termed microsatellite instability (MSI) phenotype. MSI is the conse- C.N.A. was funded by a grant of the Dr. Mildred- quence of failure of the DNA mismatch repair (MMR) system to faithfully correct errors Scheel-Stiftung, Germany. The contents of this occurring during DNA replication. MSI in HNPCC is mainly caused by germline muta- manuscript are solely the responsibility of the tions in two major MMR genes hMLH1 and hMSH2, and to a lesser extent by mutations authors and do not necessarily represent the official views of the National Cancer Institute. For further in hMSH6, hPMS2 and hMLH3. Chromosomal abnormalities are infrequent in MSI-H information regarding who participated in this cancers, and the tumor cells are most commonly diploid. Of all sporadic CRCs, 10–15% study, please see table on p. 78. www.landesbioscience.com Cancer Biology & Therapy 73 © 2004 Landes Bioscience. Not for distribution. THE MSI PHENOTYPE OF COLORECTAL CANCER possess the MSI-H phenotype. It has been demonstrated that the MATERIALS AND METHODS MMR defect in most of these tumors is caused by de novo methylation 7 Tissue Selection and DNA Extraction. A total of 173 sporadic CRC as of the hMLH1 promoter. Only MSI-H sporadic colon tumors were well as corresponding normal mucosa were collected from CALGB. For the shown to demonstrate extensive hMLH1 promoter methylation, further correlation of MSI-H tumors with hMLH1 methylation status and while a minor fraction of MSI-L and MSS cancers appear to be hMLH1 and hMSH2 protein expression, 18 formerly characterized MSI-H methylated at the hMLH1 promoter. Promoter methylation causes cancers from UCSD were included into the analyses. Five micrometer transcriptional silencing of the hMLH1 gene resulting in loss of thick serial sections from the paraffin-embedded matched normal and neo- protein expression. plastic primary tissues were stained with H&E, and representative normal Tumors progressing through the mutator or the suppressor path- and tumor regions were identified by microscopic examination. The refer- way differ in respect to their molecular and clinico-pathological fea- ence slide was compared to an unstained slide and tumor areas and adjacent tures. It has been reported that MSS/MSI-L tumors, in contrast to normal tissues were microdissected separately. Genomic DNA was isolated MSI-H tumors, occur most commonly in the left colon and rectum from the paraffin-embedded microdomains using GeneReleaser™ (Bioventure laboratories, CA). Subsequently, the DNA samples were incu- and harbor k-ras, APC, and p53 mutations more frequently, and bated overnight at 55˚C in a lysis buffer containing proteinase K and used exhibit silencing of the DNA repair gene O-6-methylguanine DNA as a template DNA for PCR analysis after heat inactivation of proteinase K methyltransferase (O -MGMT) through promoter methyla- 10,11 for 15 minutes. tion. MSI-H tumors are predominantly located in the proximal Microsatellite Analysis. Five MSI markers were utilized, consisting of colon and are often characterized histologically by poor differentia- three dinucleotide repeats (D2S123, D5S346, D17S250) and two mononu- tion, lymphocytic infiltration and extracellular mucin production. cleotide repeats (BAT25 and BAT26). In brief, genomic DNA was ampli- Both MSI-H and MSS/MSI-L tumors appear to arise from hyper- fied and labeled with the radioisotope P by 35–40 PCR cycles with a 12,13,14 plastic polyps evolving to serrated adenomas. Despite their denaturation step at 94˚C for 1 min, an annealing step of histology, HNPCC related cancers and sporadic MSI-H cancers 0 55–62˚C for 45–60 sec, and an elongation step of 72 C for 1 min in a total tend to have a better outcome in terms of disease free and overall of 5 µl of reaction mixture (0.5 µM of each primer, 1.5 mM MgCl , 0.2 survival. In contrast, data from in vitro cell models showed that mM of each deoxynucleotide triphosphate, 0.5U of Taq-DNA polymerase, MSI-H tumors have resistance against a variety of clinically used and 0.5 µl of [γ-P]dATP 10 µCi/ml). The radioisotope-labeled MSI 16-18 chemotherapeutic drugs. These features necessitate to accurately sequences were visualized by denaturing gel electrophoresis and autoradiog- raphy. Each PCR was repeated twice to ensure the reproducibility of results diagnose and distinguish cancers among the mutator and the sup- in cases where the band shifts were not clearly informative in the first pressor pathways for future evaluations and individualized treatment attempt. Tumors with a shift in at least two of the five markers were classi- strategies. fied as being MSI-H. MSI-L was defined as a shift in only one of the five In the recent years, the “gold standard” in diagnosing MSI-H markers. Tumors not showing allelic shifts were termed MSS. The scoring cancers has been MSI testing using standard PCR-methods. Several was undertaken independently by two authors (C.N.A. and A.G.) as studies have evaluated various MSI markers with different degrees of described previously. 19,20 sensitivity and specificity in their ability to diagnose MSI. In hMLH1 MSP. Methylation status of the hMLH1 promoter was deter- 1998, five “Bethesda MSI markers” were recommended by a mined by MSP. The DNA methylation pattern within the CpG islands of National Cancer Institute workshop on MSI, which are considered the promoter was determined by chemical modification of unmethylated to be able to detect MSI-H cancers with high reliability. hMLH1 but not the methylated cytosines to uracil and subsequent PCR using spe- expression determined by immunohistochemistry (IHC) has been cific primers for either methylated or the modified unmethylated DNA. compared to MSI testing in sporadic CRC and HNPCC, and a DNA (1µg) in a volume of 50 µl was denatured by NaOH for 10 min at correlation was found in detecting the loss of hMLH1 expression 37˚C. Thirty microliters of 10 mM hydroquinone (Sigma) and 520 µl of 3 22,23,24 and MSI-H. However, these strategies could not exclude the M sodium bisulfite (Sigma) at pH 5.5 were added, and samples were incu- bated at 50˚C for 16–18 h. The modified DNA was purified using the possibility of missing some cases of MSI-H cancers when IHC was Wizard DNA purification resin (Promega, Madison, WI) according to the used as a solitary technique. Recently, much emphasis has also been manufacturer’s specifications. DNA was further treated with NaOH for 10 placed on studying hMLH1 methylation in CRCs. Methylation min at room temperature and precipitated with ethanol at -80˚C. The spe- specific PCR (MSP) appears to be the method of choice to reliably 7,25 cific primers for the methylated and unmethylated MSP were as follows: study the methylation status of different gene promoters. Several hMLH1-M 5’-ACGTAGACGTTTTATTAGGGTCGC-3’ (forward), 5’- studies proved that methylation of the hMLH1 promoter strongly CCTCATCGTAACTACCCGCG-3’ (backward); hMLH1-U 5’-TTTTG- correlates with the loss of its protein expression. Despite the use of ATGTAGATGTTTTATTAGGGTTGT-3’ (forward), 5’-ACCACCTCAT- all these methods to distinguish MSI-H cancers from other sporadic CATAACTACCCACA 3’ (backward). The PCR mixture contained 1x PCR CRCs, there is no consensus opinion as to which strategy would be buffer ((NH ) SO 16.6 mM, Tris pH 8.8 67 mM, MgCl 6.7 mM, β- 4 2 4 2 most specific and efficient for defining the MSI-H phenotype. mercaptoethanol 10 mM), 0.2 mM of each dNTP, primers (500 ng each), We therefore intended to investigate the correlation among 0.2 U Taq-polymerase (Gibco) and 50 ng bisulfite-modified or unmodified conventional MSI testing, protein expression of the two major DNA. PCR reactions were hot-started at 95˚C for 5 min. Amplification was MMR genes hMLH1 and hMSH2 and the promoter methylation performed for 35 cycles (30 sec at 95˚C, 30 sec at 64˚C, 30 sec at 72˚C) status of hMLH1 in sporadic CRC to define the MSI phenotype. In followed by a final extension for 10 min at 72˚C. The PCR product was an ideal scenario, MSI-H cancers would lack hMLH1 expression electrophoresed on a 2.5 % agarose gel, stained with 0.5 µg/ml ethidium and demonstrate hMLH1 promoter methylation, which would bromide, and visualized under UV-illumination. Immunohistochemical Analysis. Immunohistochemical staining was facilitate screening methods by simply using standard and cost effec- performed using mouse monoclonal antibodies against hMLH1 (clone tive MSP techniques for categorizing CRC according to their pathway G168-15, 0.5mg/ml, PharMingen, San Diego, CA) and hMSH2 (clone of origin. FE11, 0.1mg/ml, Oncogene Research Products, Boston, MA). Sections (5 µm) of formalin-fixed, paraffin-embedded tissue were picked up on charged slides and dried overnight at 37˚C. Immunohistochemical staining was manually performed using the avidin-biotin complex (ABC) method. 74 Cancer Biology & Therapy 2004; Vol. 3 Issue 1 © 2004 Landes Bioscience. Not for distribution. THE MSI PHENOTYPE OF COLORECTAL CANCER Table 1 DISTRIBUTION OF THE MSI PHENOTYPE MSS MSI-L MSI-H tumors (n=173) (n=111) (n=35) (n=27) 64% 20% 16% Briefly, sections were deparaffinized, and endogenous peroxidase blocked using 0.3% H O in methanol. Heat-induced antigen retrieval was per- 2 2 formed in 10mM critrate buffer (pH 6.0) in a microwave oven for 15 min. Sections were incubated at 37˚C for 1 hour with primary antibody and avidin-biotin complex (Histostain-Plus Bulk Kits, Zymed Laboratories Inc., Figure 1. MSP of the hMLH1 promoter region in sporadic CRCs. Bisulfite San Francisco, CA). Sections were developed using the chromogen 3-amino- treated DNA was amplified with hMLH1 methylated and unmethylated spe- 9-ethylcarbazole, counterstained with hematoxylin, and mounted in aqueous cific primers. The PCR product indicated by U represents an unmethylated medium. Negative control sections were treated in an identical manner with allele, while the PCR product indicated by M represents a methylated allele. the exception of omission of the primary antibody. Normal colonic gland H2O represents a blank control, T1 is completely methylated, T2 is unmethy- epithelium served as the internal positive control for both antibodies. The lated and T3 carries both a methylated and an unmethylated allele. The immunohistochemical staining for hMLH1 and hMSH2 was scored by two lower bands represent nonspecific PCR products. independent observers (CC and VM). The normal staining pattern for both antibodies is nuclear. Cancers with complete loss of the nuclear staining (p<0.0001). Of the methylated MSI-H cases, 6/14 (43%) expressed compared to normal tissue were scored as negative. In cases of disagreement, hMLH1 protein while 5/6 (83%) of the unmethylated MSI-H were positive the slides were reviewed, and a consensus view was achieved. Staining for for hMLH1 staining. Sensitivity of the immunostaining in detecting MSI- hMLH1 expression analysis was attempted in all tumors but was successful H tumors was 45% and specificity was 99%, respectively. in 85 of 111 MSS tumors, 23 of 35 MSI-L tumors and 20 of 27 MSI-H tumors. MSH2 immunostaining was attempted and successful in all We further correlated hMLH1 expression with the hMLH1 promoter tumors. Cases that had inadequate internal control tissue or inadequate methylation status of the MSI-H tumors. Eighteen UCSD MSI-H tumors tumor in the section or in which the staining was technically inadequate were included in this analysis. Of the MSI-H cases, 4 lacked expression of were eliminated. the hMSH2 protein. These 4 tumors were not included in this analysis, Statistical Analysis. Fisher’s exact test and the chi-square test were used because they were considered to belong to the HNPCC spectrum. Of 45 as appropriate to test the associations between hMLH1 and hMSH2 protein total MSI-H tumors, 14 were missing data on one or both of hMLH1 expression and MSI status and to test associations within the MSI-H sub- methylation and protein expression and 4 were negative on hMSH2. The 27 group. The analyses involving MSI status were performed on 173 CALGB remaining MSI-H tumors were included in this analysis. tumor samples. Analyses within the MSI-H subgroup included data from an Of 15 MSI-H cancers with loss of hMLH1 expression, 14 (93%) were additional 18 UCSD tumor samples. Sample sizes in each analysis varied also methylated while 42% MSI-H cancers (5/12) with positive immunos- according to the numbers of samples analysed for each of MSI status, taining for hMLH1 were methylated at the hMLH1 promoter (p = 0.009) hMLH1 and hMSH2 protein expression and hMLH1 methylation. All (Table 3). We thus were able to demonstrate the close relationship of statistical analyses were performed at the CALGB statistical center. hMLH1 protein expression with the hMLH1 promoter methylation status. Ability of MSI Markers to Determine the MSI Phenotype. Previous studies have reported a high accuracy of specific MSI markers in detecting RESULTS the MSI-H phenotype, especially for the highly monomorphic mononu- MSI Status, Correlation with hMLH1 Promoter Methylation and cleotide marker BAT26. We compared the sensitivity and specificity of MMR-Gene Expression. Of the 173 tumors from CALGB, 111 (64%) each individual marker of the Bethesda panel for its ability to define the were MSS, 35 (20%) were MSI-L and 27 (16%) were MSI-H (Table 1). In MSI-H (n = 45) phenotype based on all 5 markers. All 45 MSI-H tumors order to rule out further undetected MSI, we performed MSI analysis with (from CALGB and UCSD) were included in this analysis. We found that no additional mononucleotide (TGFβRII, IGFIIR, BAX, hMSH3, hMSH6), individual marker on its own was able to reliably detect the MSI-H pheno- di-nucleotide (D3S1029, D17S261, D18S64, D18S69 and D18S474) and type. Sensitivity of the individual MSI markers in detecting MSI-H was tetra-nucleotide markers (MYCL1). Even by adding these markers, the overall MSI pattern did not change. We further investigated the methylation status of the hMLH1 promoter. We were able to successfully perform the MSP assay in 20 MSI-H and 27 MSI-L cancers (Fig. 1). Thirty-five MSS tumors were non-randomly selected for MSP analysis of which 34 were successful. As expected, most of the MSS (28/34, 82%) and MSI-L (22/27, 81%) cancers were unmethylated at the hMLH1 promoter, while 6 of 20 (30%) MSI-H cancers were unmethylated (p<0.0001) (Fig. 2). Subsequently, we performed expression analysis of the MMR proteins hMLH1 and hMSH2 in the CALGB tumor cohort (Table 2, Fig. 3). Of 108 MSS and MSI-L cancers investigated, 107 (99%) expressed hMLH1. All but one tumor were unmethylated. Of the MSI-H cancers, 11/20 (55%) expressed hMLH1 Table 2 hMLH1 EXPRESSION IN ALL TUMORS INVESTIGATED MSS MSI-L MSI-H tumors (n = 128) (n = 84) (n = 24) (n = 20) Figure 2. Methylation status according to the degree of MSI. The hMLH1 99% 100% 55% promoter methylation was investigated by MSP in each tumor group. www.landesbioscience.com Cancer Biology & Therapy 75 © 2004 Landes Bioscience. Not for distribution. THE MSI PHENOTYPE OF COLORECTAL CANCER Table 3 CORRELATION OF MMR GENE EXPRESSION AND METHYLATION STATUS IN MSI-H CANCERS (INCLUDING CALGB AND UCSD CASES) Tumors Methylated Unmethylated Total Lack of hMLH1 14 (93%) 1 (7%) 15 expression hMLH1 expression 5 (42%) 7 (58%) 12 Total 19 (70%) 8 (30%) 27 this, an important issue still remains as to how the difference in sen- sitivity of immunohistochemistry in MSI-H cancers demonstrated in our and previous studies can be best explained. One issue is the Figure 3. hMLH1 expression. hMLH1 protein expression was investigated in definition of the MSI phenotype itself. It has been convincingly all tumor groups by IHC. shown that the MSI-H phenotype is correlated in most cases with aberrant methylation of the hMLH1 promoter which influences the highest for the dinucleotide markers D17S250 and D5S346 with 76% and transcriptional activity of the gene. We therefore investigated the 73%, respectively. Specificity was best for BAT26 and BAT25 with 99% and promoter methylation status of our entire tumor cohort with the 95%, respectively (Table 4). MSI status. We found that most MSS/MSI-L cancers had an unmethylated and, presumably, transcriptionally active hMLH1 DISCUSSION promoter. In 99% of these cases, the tumors were shown to express hMLH1 and hMSH2 proteins by immunohistochemistry. However, Molecular classification of colorectal cancer based on its molecu- lar features has important implications regarding prognosis and 18% of the MSS and 19% of the MSI-L cancers showed methyla- tion. Surprisingly, only 70% of the MSI-H cancers demonstrated might influence future treatment strategies. It has been shown that MSI-H sporadic colon cancers have in vitro resistance to common- promoter hypermethylation. Correlating the immunohistochemistry findings and the promoter methylation status in MSI-H cancers, we ly used chemotherapeutic drugs compared to cancers with the MSS and MSI-L phenotype. It remains to be determined if this is also found a strong association between negative immunohistochemistry and promoter methylation. However, the association between relevant in vivo. Up to now, only one retrospective study showed that 5-FU based chemotherapy might influence overall and disease hMLH1 expression and promoter methylation status was not absolute, as 7% of the MSI-H tumors lacking hMLH1 expression free survival negatively in patients with stage II and III CRCs. The MSI-H phenotype arises through the defects of the human were unmethylated and 42% of the hMLH1 expressing neoplasms were methylated. Although it would have been interesting to obtain MMR system, notably through transcriptional silencing of the hMLH1 gene by aberrant methylation of its promoter. Promoter data on DNA sequencing on those MSI-H cases that retained hMLH1 or hMSH2 expression in order to investigate if those methylation has been demonstrated to inhibit hMLH1 protein expression. So far, MSI-H cancers have been diagnosed by MSI tumors harbor somatic missense mutations that inactivate protein function. Unfortunately the amount of tumor DNA available did testing using a panel of consensus MSI markers. By definition, tumors with band shifts in 30–40% or more of the tested MSI mark- not allow us to perform such analysis for the current study. All MSI-H tumors in this study were defined using Bethesda ers are scored as MSI-H. However, no study has yet systematically compared the correlation of MSI-H sporadic colon cancers diagnosed criteria. Our findings raise two issues: 1) whether this definition cor- rectly categorizes all MSI tumors; and 2) whether all tumors with the by conventional MSI testing with the hMLH1 promoter methylation status and hMLH1 protein expression, respectively. It seems feasible MSI-H phenotype develop through the same molecular mecha- nisms. It has been uniformly assumed that hMLH1 promoter that searching for cancers with a silenced hMLH1 gene by promoter methylation could all be performed by simply employing the MSP methylation is responsible for transcriptional silencing and is related to the MSI-H phenotype. In our study, only 70% of the MSI-H technique instead of PCR testing with 5 standard MSI markers. With this strategy, screening for these cancers could be facilitated. cancers are hypermethylated and 45% of the MSI-H cancers expressed hMLH1. Some of these cases can be explained by defects In our study we assessed the benefits of MSI testing, hMLH1 promoter methylation analysis and hMLH1 protein expression for of other MMR genes such as hMSH2 (4 cases in this study). Presumably, these were not sporadic cancers but rather belonged to the determination of sporadic CRC with the MSI-H pheno- 23,29,30 type. First, we demonstrated the expected frequency of the MSI phenotype in sporadic colorectal cancer with 16% of all cancers Table 4 SENSITIVITY AND SPECIFICITY OF INDIVIDUAL MSI MARKER being MSI-H. Interestingly, only 45% of these cancers showed absence of hMLH1 expression. Earlier studies have estimated sensi- Sensitivity Specificity tivity between 75% and 100% for negative immunostaining and the MSI-H phenotype. However, in our study only 1 of 108 MSS/MSI-L D2S123 69% 85% cancers did not express hMLH1 protein suggesting a high specificity of D5S346 76% 79% MMR gene expression in non-MSI-H cancers. This is in accordance D17S250 73% 85% with others, all showing a specificity of 100% of immunohisto- BAT25 67% 95% chemistry in correctly diagnosing the MSS phenotype in cancers 22,23,29-31 BAT26 71% 99% with positive hMLH1 and hMSH2 expression. Despite 76 Cancer Biology & Therapy 2004; Vol. 3 Issue 1 © 2004 Landes Bioscience. Not for distribution. THE MSI PHENOTYPE OF COLORECTAL CANCER 8. Laiho P, Launonen V, Lahermo P, Esteller M, Guo M, Herman JG, et al. Low-level the familial tumor syndrome HNPCC. Our findings are in agree- microsatellite instability in most colorectal carcinomas. Cancer Res 2002; 62:1166-70. ment with a recent study showing that only 47% of MSI positive 9. Kane MF, Loda M, Gaida GM, Lipman J, Mishra R, Goldman H, et al. Methylation of the sporadic colorectal tumors with absent hMLH1 expression were also hMLH1 promoter correlates with lack of expression of hMLH1 in sporadic colon tumors and mismatch repair-defective human tumor cell lines. Cancer Res 1997; 57:808-11. hypermethylated at the hMLH1 promoter. It is not known what 10. Whitehall VL, Walsh MD, Young J, Leggett BA, Jass JR. Methylation of O-6-methylgua- role other MMR or DNA repair genes play in the development of nine DNA methyltransferase characterizes a subset of colorectal cancer with low-level DNA the MSI-H phenotype. So far, it has not been demonstrated that any microsatellite instability. Cancer Res 2001; 61:827-30. 11. Jass JR, Biden KG, Cummings MC, Simms LA, Walsh M, Schoch E, et al. Characterisation of the known MMR genes plays a major role in causing the MSI-H 6 of a subtype of colorectal cancer combining features of the suppressor and mild mutator phenotype in sporadic colorectal cancer. Only the O -MGMT gene, pathways. J Clin Pathol 1999; 52:455-60. when methylated and transcriptionally silenced, has been clearly 12. Chao A, Gilliland F, Willman C, Joste N, Chen IM, Stone N, et al. Patient and tumor char- acteristics of colon cancers with microsatellite instability: A population-based study. Cancer linked to cancers with the MSI-L, but not the MSI-H, phenotype. Epidemiol Biomarkers Prev 2000; 9:539-44. The question of how the MSI phenotype should be truly defined 13. Hawkins NJ, Ward RL. Sporadic colorectal cancers with microsatellite instability and their remains unanswered. We demonstrated that most cancers which did possible origin in hyperplastic polyps and serrated adenomas. J Natl Cancer Inst 2001; 93:1307-13. not show immunoreactivity for hMLH1 or hMSH2 were also 14. Jass JR, Iino H, Ruszkiewicz A, Painter D, Solomon MJ, Koorey DJ, et al. Neoplastic pro- methylated for hMLH1. Conversely, most of the hMLH1 and gression occurs through mutator pathways in hyperplastic polyposis of the colorectum. Gut hMSH2 expressing MSI-H cancers were unmethylated. MSI-H was 2000; 47:43-9. diagnosed using the reference panel of 5 MSI standard markers and 15. Ribic CM, Sargent DJ, Moore MJ, Thibodeau SN, French AJ, Goldberg RM, et al. Tumor microsatellite-instability status as a predictor of benefit from fluorouracil-based adjuvant no significant difference was evident between the instability pattern chemotherapy for colon cancer. N Engl J Med 2003; 349:247-57. of hMLH1 expressing and non-expressing cancers with respect to 16. Aebi S, Kurdi-Haidar B, Gordon R, Cenni B, Zheng H, Fink D, et al. Loss of DNA mis- the markers tested. match repair in acquired resistance to cisplatin. Cancer Res 1996; 56:3087-90. 17. Carethers JM, Chauhan DP, Fink D, Nebel S, Bresalier RS, Howell SB, et al. Mismatch repair All MSI markers used in the study had a high specificity but a proficiency and in vitro response to 5-fluorouracil. Gastroenterology 1999; 117:123-31. rather low sensitivity, suggesting that no marker on its own was able 18. Arnold CN, Goel A, Boland CR. Role of hMLH1 hypermethylation in drug resistance to to reliably detect all MSI-H cancers. However, when performing 5-fluorouracil in colorectal cancer cell lines. Int J Cancer 2003; 106:66-73. MSI analysis comparing MSI-H and hMLH1 hypermethylated or 19. Bocker T, Diermann J, Friedl W, Gebert J, Holinski-Feder E, Karner-Hanusch J, et al. Microsatellite instability analysis: A multicenter study for reliability and quality control. MSI-H and unmethylated cancers, all markers but the dinucleotide Cancer Res 1997; 57:4739-43. repeat D2S123 had a high detection rate up to 90% for this selection 20. Dietmaier W, Wallinger S, Bocker T, Kullmann F, Fishel R, Ruschoff J. Diagnostic microsatellite instability: Definition and correlation with mismatch repair protein expres- of cancers. MSI-H cancers might originate from different pathways, sion. Cancer Res 1997; 57:4749-56. one being caused by silencing of the hMLH1 gene and others by yet 21. Boland CR, Thibodeau SN, Hamilton SR, Sidransky D, Eshleman JR, Burt RW, et al. A unrecognized causes. national cancer institute workshop on microsatellite instability for cancer detection and The issue which should be further addressed in future studies is familial predisposition: Development of international criteria for the determination of microsatellite instability in colorectal cancer. Cancer Res 1998; 58:5248-57. the extent and amount of promoter methylation within the hMLH1 22. Chaves P, Cruz C, Lage P, Claro I, Cravo M, Leitao CN, et al. Immunohistochemical detec- promoter causing transcriptional silencing of the gene. Earlier studies tion of mismatch repair gene proteins as a useful tool for the identification of colorectal car- cinoma with the mutator phenotype. J Pathol 2000; 191:355-60. reported a significant concordance between methylation of a 5’ 23. Lindor NM, Burgart LJ, Leontovich O, Goldberg RM, Cunningham JM, Sargent DJ, et promoter region (also called region A) with the MSI-H phenotype. al. Immunohistochemistry versus microsatellite instability testing in phenotyping colorec- Others reported a further downstream area of the promoter (region C) tal tumors. J Clin Oncol 2002; 20:1043-8. being more closely related to MSI-H cancers. However, their study 24. Salahshor S, Koelble K, Rubio C, Lindblom A. Microsatellite instability and hMLH1 and hMSH2 expression analysis in familial and sporadic colorectal cancer. Lab Invest 2001; was performed in various colorectal tumor cell lines and may not be 81:535-41. directly extrapolated to the in vivo setting. One recent study exam- 25. Menigatti M, Di Gregorio C, Borghi F, Sala E, Scarselli A, Pedroni M, et al. Methylation ined the methylation status of the whole hMLH1 promoter and pattern of different regions of the MLH1 promoter and silencing of gene expression in hereditary and sporadic colorectal cancer. Genes Chromosomes Cancer 2001; 31:357-61. concluded that MSI-H cancers usually possess a full methylation 26. Goel A, Arnold CN, Niedzwiecki D, Chang DK, Ricciardiello L, Carethers JM, et al. pattern of the whole promoter rather than a partial one. We Characterization of sporadic colon cancer by patterns of genomic instability. Cancer Res performed methylation analysis of the promoter region A in agreement 2003; 63:1608-14. 27. Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB. Methylation-specific PCR: A with most other studies. novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA 1996; From the findings of this study it seems feasible to continue MSI 93:9821-6. testing with standard MSI analysis. Promoter methylation analysis 28. Zhou XP, Hoang JM, Li YJ, Seruca R, Carneiro F, Sobrinho-Simoes M, et al. and staining for MMR gene expression are not yet capable by them- Determination of the replication error phenotype in human tumors without the require- ment for matching normal DNA by analysis of mononucleotide repeat microsatellites. selves to distinguish MSI-H from MSS/MSI-L cancers. However, Genes Chromosomes Cancer 1998; 21:101-7. they are able to complement the screening. 29. Cawkwell L, Gray S, Murgatroyd H, Sutherland F, Haine L, Longfellow M, et al. Choice of management strategy for colorectal cancer based on a diagnostic immunohistochemical References test for defective mismatch repair. Gut 1999; 45:409-15. 1. Fearon ER, Vogelstein B. A genetic model for colorectal tumorigenesis. Cell 1990; 61:759-67. 30. Terdiman JP, Gum Jr JR, Conrad PG, Miller GA, Weinberg V, Crawley SC, et al. Efficient 2. Aaltonen LA, Peltomaki P, Leach FS, Sistonen P, Pylkkanen L, Mecklin JP, et al. Clues to detection of hereditary nonpolyposis colorectal cancer gene carriers by screening for tumor the pathogenesis of familial colorectal cancer. Science 1993; 260:812-6. microsatellite instability before germline genetic testing. Gastroenterology 2001; 120:21-30. 3. Ionov Y, Peinado MA, Malkhosyan S, Shibata D, Perucho M. Ubiquitous somatic muta- 31. Marcus VA, Madlensky L, Gryfe R, Kim H, So K, Millar A et al. Immunohistochemistry tions in simple repeated sequences reveal a new mechanism for colonic carcinogenesis. for hMLH1 and hMSH2: A practical test for DNA mismatch repair-deficient tumors. Am Nature 1993; 363:558-61. J Surg Pathol 1999; 23:1248-55. 4. Thibodeau SN, Bren G, Schaid D. Microsatellite instability in cancer of the proximal 32. Deng G, Chen A, Hong J, Chae HS, Kim YS. Methylation of CpG in a small region of the colon. Science 1993; 260:816-9. hMLH1 promoter invariably correlates with the absence of gene expression. Cancer Res 5. Marra G, Boland CR. DNA repair and colorectal cancer. Gastroenterol Clin North Am 1999; 59:2029-33. 1996; 25:755-72. 33. Goel A, Arnold CN, Boland CR. Multistep progression of colorectal cancer in the setting 6. Boland CR. Molecular genetics of hereditary nonpolyposis colorectal cancer. Ann N Y of microsatellite instability: New details and novel insights. Gastroenterology 2001; Acad Sci 2000; 910:50-9. 121:1497-502. 7. Herman JG, Umar A, Polyak K, Graff JR, Ahuja N, Issa JP, et al. Incidence and function- 34. Miyakura Y, Sugano K, Konishi F, Ichikawa A, Maekawa M, Shitoh K, et al. Extensive al consequences of hMLH1 promoter hypermethylation in colorectal carcinoma. Proc Natl methylation of hMLH1 promoter region predominates in proximal colon cancer with Acad Sci USA 1998; 95:6870-5. microsatellite instability. Gastroenterology 2001; 121:1300-9. www.landesbioscience.com Cancer Biology & Therapy 77 © 2004 Landes Bioscience. Not for distribution. THE MSI PHENOTYPE OF COLORECTAL CANCER THE FOLLOWING INSTITUTIONS PARTICIPATED IN THE STUDY Institution Name Location PI Grant # CALGB Statistical Office Durham, NC Stephen George, Ph.D supported by CA33601 Dana Farber Canc78er Institute Boston, MA George P. Canellos, MD supported by CA32291 Dartmouth Medical School-Norris Cotton Lebanon, NH Marc Ernstoff MD supported by CA04326 Cncr Ctr Massachusetts General Hospital Boston, MA Michael L. Grossbard, M.D. supported by CA12449 Mount Sinai School of Medicine New York, NY Lewis Silverman, MD supported by CA04457 Rhode Island Hospital Providence, RI William Sikov, M.D. supported by CA08025 Roswell Park Cancer Institute Buffalo, NY Ellis Levine, M.D. supported by CA02599 Southeast Cancer Control Consortium Inc. Goldsboro, NC James N. Atkins, M.D. supported by CA45808 SUNY Upstate Medical University Syracuse, NY Stephen L. Graziano, M.D. supported by CA21060 The Ohio State University Columbus, OH Clara D. Bloomfield, MD supported by CA77658 University of California at San Diego San Diego, CA Stephen Seagren, MD supported by CA11789 University of California at San Francisco San Francisco, CA Alan Venook, MD supported by CA60138 University of Chicago Medical Center Chicago, IL Gini Fleming, M.D. supported by CA41287 University of Illinois at Chicago Chicago, IL David Gustin, M.D. supported by CA74811 University of Iowa Iowa City, IA Gerald Clamon, MD supported by CA47642 University of Maryland Cancer Center Baltimore, MD David Van Echo, M.D. supported by CA31983 University of Massachusetts Medical Center Worcester, MA Mary Ellen Taplin, M.D. supported by CA37135 University of Minnesota Minneapolis, MN Bruce A Peterson, M.D. supported by CA16450 University of Missouri/Ellis Fischel Cancer Columbia, MO Michael C Perry, M.D. supported by CA12046 Center University of North Carolina at Chapel Hill Chapel Hill, NC Thomas C. Shea, M.D. supported by CA47559 University of Tennessee Memphis Memphis, TN Harvey B. Niell, M.D. supported by CA47555 Wake Forest University School of Medicine Winston-Salem, NC David D. Hurd, MD supported by CA03927 Walter Reed Army Medical Center Washington, DC John C. Byrd, M.D. supported by CA26806 78 Cancer Biology & Therapy 2004; Vol. 3 Issue 1 © 2004 Landes Bioscience. Not for distribution. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Cancer Biology & Therapy Taylor & Francis

Evaluation of Microsatellite Instability, hMLH1 Expression and hMLH1 Promoter Hypermethylation in Defining the MSI Phenotype of Colorectal Cancer

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Taylor & Francis
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Copyright © 2003 Landes Bioscience
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1555-8576
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1538-4047
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10.4161/cbt.3.1.590
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[Cancer Biology & Therapy 3:1, 73-78, January 2004]; ©2004 Landes Bioscience Research Paper Evaluation of Microsatellite Instability, hMLH1 Expression and hMLH1 Promoter Hypermethylation in Defining the MSI Phenotype of Colorectal Cancer 1,† ABSTRACT Christian N. Arnold 1,† Introduction: About 15% of all colorectal cancers (CRCs) demonstrate high levels of Ajay Goel microsatellite instability (MSI-H) and are currently best identified by molecular analysis of Carolyn Compton microsatellite markers. Most sporadic CRCs with MSI-H are known to be associated with the methylation of the hMLH1 promoter. Promoter methylation coincided with lack of Victoria Marcus hMLH1 expression. We aimed to investigate the association between MSI status, hMLH1 Donna Niedzwiecki protein expression and methylation status of the hMLH1 promoter, and to determine the use- fulness of each method in defining the MSI phenotype in sporadic CRCs. Materials and Jeannette M. Dowell Methods: CRCs from 173 patients from the Cancer and Leukemia Group B (CALGB) were Linda Wasserman assessed for their MSI status. An additional cohort of 18 MSI-H tumors from the University of California San Diego (UCSD) was included in the analysis of the MSI-H subgroup. Toru Inoue MSI testing was performed by PCR using five standard MSI markers. hMLH1 promoter Robert J. Mayer analysis was investigated by methylation specific PCR (MSP), and expression of the MMR 5 genes hMLH1 and hMSH2 was examined by immunohistochemistry (IHC). Results: Of the Monica M Bertagnolli 173 CALGB tumors, 111 (64%) were MSS, 35 (20%) were MSI-L and 27 (16%) MSI-H, 1,6, C. Richard Boland * respectively. Data on hMLH1 protein expression, hMSH2 protein expression and hMLH1 methylation are available on 128, 173 and 81 of these tumors, respectively. Presence of Department of Medicine and Cancer Center; University of California San Diego; La hMLH1 and hMSH2 protein expression was significantly associated with MSI status. Four Jolla, California USA of 45 (8.9%) MSI-H tumors and 0 of 146 (0%) MSS/MSI-L tumors did not express hMSH2 Department of Pathology; McGill University; Montreal, Quebec, Canada (p = 0.0028). hMLH1 protein expression was present in 107 of 108 (99%) MSS and CALGB Statistical Center; Duke University; Durham, North Carolina USA MSI-L tumors versus 11 of 20 (55%) MSI-H tumors (p < 0.0001). Of 61 MSS and MSI-L 4 cancers studied for methylation, 11 (18%) were methylated at the hMLH1 promoter Department of Medicine; Dana Farber Cancer Institute; Boston, Massachusetts USA whereas 14 of 20 (70%) MSI-H cancers were methylated (p = 0.0001). In 27 MSI-H Brigham and Women’s Hospital; Boston, Massachusetts USA tumors studied for hMLH1 protein expression and methylation, 93% of tumors with loss San Diego Veteran Affairs Medical Center; La Jolla, California USA of expression (93%) were also methylated while 42% (5/12) with positive immunostain- ing for hMLH1 were methylated at the hMLH1 promoter (p = 0.009). Conclusions: These authors contributed equally to this study Promoter methylation and hMLH1 expression are significantly associated with the MSI-H *Correspondence to: C. Richard Boland; Baylor University Medical Center; 2 phenotype in CRC. Promoter methylation analysis provides a useful means to screen for Hoblitzelle, 3500 Gaston Avenue; Dallas, Texas USA; Tel.: 214.820.2692; Fax: 214.818.9292; Email: RickBo@BaylorHealth.Edu MSI-H tumors. Our data further suggests that hMLH1 promoter methylation analysis alone cannot replace MSI testing, as a significant number of MSI-H tumors could be potentially Received 07/29/03; Accepted 09/05/03 overseen by such an approach. We suggest that phenotypic evaluation of CRC is performed Previously published online as a Cancer Biology & Therapy E-publication: most reliably with MSI testing, although expression analysis and investigation of the promoter http://www.landesbioscience.com/journals/cbt/abstract.php?id=590 methylation status may complement the screening process. KEY WORDS hMLH1, microsatellite instability, promoter methylation, colorectal cancer, immunohistochem- INTRODUCTION istry CRC pathogenesis proceeds through two well defined pathways of genomic instability, ACKNOWLEDGEMENTS termed as the suppressor and mutator pathways. These pathways are characterized by successive accumulation of genetic events in neoplastic cells as a function of time. In most The research for CALGB study 9865 was sup- human neoplasias, the first mechanism is observed at the chromosomal level and leads to ported, in part, by grants from the National Cancer Institute (CA31946) to the Cancer and Leukemia gene alterations by chromosomal gains and losses, accompanied by mutations at specific Group B (Richard L. Schilsky, Chairman). This tumor suppressor genes and oncogenes. The second mechanism was first described in work was also sponsored by a grant from the NIH 1993 by observing size variations of short repetitive DNA sequences in tumor DNA, (RO1-CA 72851) to C.R.B and in part by an 2-4 termed microsatellites. It was soon linked to hereditary non-polyposis colon cancer American Cancer Society-IRG award to A.G. (HNPCC) and termed microsatellite instability (MSI) phenotype. MSI is the conse- C.N.A. was funded by a grant of the Dr. Mildred- quence of failure of the DNA mismatch repair (MMR) system to faithfully correct errors Scheel-Stiftung, Germany. The contents of this occurring during DNA replication. MSI in HNPCC is mainly caused by germline muta- manuscript are solely the responsibility of the tions in two major MMR genes hMLH1 and hMSH2, and to a lesser extent by mutations authors and do not necessarily represent the official views of the National Cancer Institute. For further in hMSH6, hPMS2 and hMLH3. Chromosomal abnormalities are infrequent in MSI-H information regarding who participated in this cancers, and the tumor cells are most commonly diploid. Of all sporadic CRCs, 10–15% study, please see table on p. 78. www.landesbioscience.com Cancer Biology & Therapy 73 © 2004 Landes Bioscience. Not for distribution. THE MSI PHENOTYPE OF COLORECTAL CANCER possess the MSI-H phenotype. It has been demonstrated that the MATERIALS AND METHODS MMR defect in most of these tumors is caused by de novo methylation 7 Tissue Selection and DNA Extraction. A total of 173 sporadic CRC as of the hMLH1 promoter. Only MSI-H sporadic colon tumors were well as corresponding normal mucosa were collected from CALGB. For the shown to demonstrate extensive hMLH1 promoter methylation, further correlation of MSI-H tumors with hMLH1 methylation status and while a minor fraction of MSI-L and MSS cancers appear to be hMLH1 and hMSH2 protein expression, 18 formerly characterized MSI-H methylated at the hMLH1 promoter. Promoter methylation causes cancers from UCSD were included into the analyses. Five micrometer transcriptional silencing of the hMLH1 gene resulting in loss of thick serial sections from the paraffin-embedded matched normal and neo- protein expression. plastic primary tissues were stained with H&E, and representative normal Tumors progressing through the mutator or the suppressor path- and tumor regions were identified by microscopic examination. The refer- way differ in respect to their molecular and clinico-pathological fea- ence slide was compared to an unstained slide and tumor areas and adjacent tures. It has been reported that MSS/MSI-L tumors, in contrast to normal tissues were microdissected separately. Genomic DNA was isolated MSI-H tumors, occur most commonly in the left colon and rectum from the paraffin-embedded microdomains using GeneReleaser™ (Bioventure laboratories, CA). Subsequently, the DNA samples were incu- and harbor k-ras, APC, and p53 mutations more frequently, and bated overnight at 55˚C in a lysis buffer containing proteinase K and used exhibit silencing of the DNA repair gene O-6-methylguanine DNA as a template DNA for PCR analysis after heat inactivation of proteinase K methyltransferase (O -MGMT) through promoter methyla- 10,11 for 15 minutes. tion. MSI-H tumors are predominantly located in the proximal Microsatellite Analysis. Five MSI markers were utilized, consisting of colon and are often characterized histologically by poor differentia- three dinucleotide repeats (D2S123, D5S346, D17S250) and two mononu- tion, lymphocytic infiltration and extracellular mucin production. cleotide repeats (BAT25 and BAT26). In brief, genomic DNA was ampli- Both MSI-H and MSS/MSI-L tumors appear to arise from hyper- fied and labeled with the radioisotope P by 35–40 PCR cycles with a 12,13,14 plastic polyps evolving to serrated adenomas. Despite their denaturation step at 94˚C for 1 min, an annealing step of histology, HNPCC related cancers and sporadic MSI-H cancers 0 55–62˚C for 45–60 sec, and an elongation step of 72 C for 1 min in a total tend to have a better outcome in terms of disease free and overall of 5 µl of reaction mixture (0.5 µM of each primer, 1.5 mM MgCl , 0.2 survival. In contrast, data from in vitro cell models showed that mM of each deoxynucleotide triphosphate, 0.5U of Taq-DNA polymerase, MSI-H tumors have resistance against a variety of clinically used and 0.5 µl of [γ-P]dATP 10 µCi/ml). The radioisotope-labeled MSI 16-18 chemotherapeutic drugs. These features necessitate to accurately sequences were visualized by denaturing gel electrophoresis and autoradiog- raphy. Each PCR was repeated twice to ensure the reproducibility of results diagnose and distinguish cancers among the mutator and the sup- in cases where the band shifts were not clearly informative in the first pressor pathways for future evaluations and individualized treatment attempt. Tumors with a shift in at least two of the five markers were classi- strategies. fied as being MSI-H. MSI-L was defined as a shift in only one of the five In the recent years, the “gold standard” in diagnosing MSI-H markers. Tumors not showing allelic shifts were termed MSS. The scoring cancers has been MSI testing using standard PCR-methods. Several was undertaken independently by two authors (C.N.A. and A.G.) as studies have evaluated various MSI markers with different degrees of described previously. 19,20 sensitivity and specificity in their ability to diagnose MSI. In hMLH1 MSP. Methylation status of the hMLH1 promoter was deter- 1998, five “Bethesda MSI markers” were recommended by a mined by MSP. The DNA methylation pattern within the CpG islands of National Cancer Institute workshop on MSI, which are considered the promoter was determined by chemical modification of unmethylated to be able to detect MSI-H cancers with high reliability. hMLH1 but not the methylated cytosines to uracil and subsequent PCR using spe- expression determined by immunohistochemistry (IHC) has been cific primers for either methylated or the modified unmethylated DNA. compared to MSI testing in sporadic CRC and HNPCC, and a DNA (1µg) in a volume of 50 µl was denatured by NaOH for 10 min at correlation was found in detecting the loss of hMLH1 expression 37˚C. Thirty microliters of 10 mM hydroquinone (Sigma) and 520 µl of 3 22,23,24 and MSI-H. However, these strategies could not exclude the M sodium bisulfite (Sigma) at pH 5.5 were added, and samples were incu- bated at 50˚C for 16–18 h. The modified DNA was purified using the possibility of missing some cases of MSI-H cancers when IHC was Wizard DNA purification resin (Promega, Madison, WI) according to the used as a solitary technique. Recently, much emphasis has also been manufacturer’s specifications. DNA was further treated with NaOH for 10 placed on studying hMLH1 methylation in CRCs. Methylation min at room temperature and precipitated with ethanol at -80˚C. The spe- specific PCR (MSP) appears to be the method of choice to reliably 7,25 cific primers for the methylated and unmethylated MSP were as follows: study the methylation status of different gene promoters. Several hMLH1-M 5’-ACGTAGACGTTTTATTAGGGTCGC-3’ (forward), 5’- studies proved that methylation of the hMLH1 promoter strongly CCTCATCGTAACTACCCGCG-3’ (backward); hMLH1-U 5’-TTTTG- correlates with the loss of its protein expression. Despite the use of ATGTAGATGTTTTATTAGGGTTGT-3’ (forward), 5’-ACCACCTCAT- all these methods to distinguish MSI-H cancers from other sporadic CATAACTACCCACA 3’ (backward). The PCR mixture contained 1x PCR CRCs, there is no consensus opinion as to which strategy would be buffer ((NH ) SO 16.6 mM, Tris pH 8.8 67 mM, MgCl 6.7 mM, β- 4 2 4 2 most specific and efficient for defining the MSI-H phenotype. mercaptoethanol 10 mM), 0.2 mM of each dNTP, primers (500 ng each), We therefore intended to investigate the correlation among 0.2 U Taq-polymerase (Gibco) and 50 ng bisulfite-modified or unmodified conventional MSI testing, protein expression of the two major DNA. PCR reactions were hot-started at 95˚C for 5 min. Amplification was MMR genes hMLH1 and hMSH2 and the promoter methylation performed for 35 cycles (30 sec at 95˚C, 30 sec at 64˚C, 30 sec at 72˚C) status of hMLH1 in sporadic CRC to define the MSI phenotype. In followed by a final extension for 10 min at 72˚C. The PCR product was an ideal scenario, MSI-H cancers would lack hMLH1 expression electrophoresed on a 2.5 % agarose gel, stained with 0.5 µg/ml ethidium and demonstrate hMLH1 promoter methylation, which would bromide, and visualized under UV-illumination. Immunohistochemical Analysis. Immunohistochemical staining was facilitate screening methods by simply using standard and cost effec- performed using mouse monoclonal antibodies against hMLH1 (clone tive MSP techniques for categorizing CRC according to their pathway G168-15, 0.5mg/ml, PharMingen, San Diego, CA) and hMSH2 (clone of origin. FE11, 0.1mg/ml, Oncogene Research Products, Boston, MA). Sections (5 µm) of formalin-fixed, paraffin-embedded tissue were picked up on charged slides and dried overnight at 37˚C. Immunohistochemical staining was manually performed using the avidin-biotin complex (ABC) method. 74 Cancer Biology & Therapy 2004; Vol. 3 Issue 1 © 2004 Landes Bioscience. Not for distribution. THE MSI PHENOTYPE OF COLORECTAL CANCER Table 1 DISTRIBUTION OF THE MSI PHENOTYPE MSS MSI-L MSI-H tumors (n=173) (n=111) (n=35) (n=27) 64% 20% 16% Briefly, sections were deparaffinized, and endogenous peroxidase blocked using 0.3% H O in methanol. Heat-induced antigen retrieval was per- 2 2 formed in 10mM critrate buffer (pH 6.0) in a microwave oven for 15 min. Sections were incubated at 37˚C for 1 hour with primary antibody and avidin-biotin complex (Histostain-Plus Bulk Kits, Zymed Laboratories Inc., Figure 1. MSP of the hMLH1 promoter region in sporadic CRCs. Bisulfite San Francisco, CA). Sections were developed using the chromogen 3-amino- treated DNA was amplified with hMLH1 methylated and unmethylated spe- 9-ethylcarbazole, counterstained with hematoxylin, and mounted in aqueous cific primers. The PCR product indicated by U represents an unmethylated medium. Negative control sections were treated in an identical manner with allele, while the PCR product indicated by M represents a methylated allele. the exception of omission of the primary antibody. Normal colonic gland H2O represents a blank control, T1 is completely methylated, T2 is unmethy- epithelium served as the internal positive control for both antibodies. The lated and T3 carries both a methylated and an unmethylated allele. The immunohistochemical staining for hMLH1 and hMSH2 was scored by two lower bands represent nonspecific PCR products. independent observers (CC and VM). The normal staining pattern for both antibodies is nuclear. Cancers with complete loss of the nuclear staining (p<0.0001). Of the methylated MSI-H cases, 6/14 (43%) expressed compared to normal tissue were scored as negative. In cases of disagreement, hMLH1 protein while 5/6 (83%) of the unmethylated MSI-H were positive the slides were reviewed, and a consensus view was achieved. Staining for for hMLH1 staining. Sensitivity of the immunostaining in detecting MSI- hMLH1 expression analysis was attempted in all tumors but was successful H tumors was 45% and specificity was 99%, respectively. in 85 of 111 MSS tumors, 23 of 35 MSI-L tumors and 20 of 27 MSI-H tumors. MSH2 immunostaining was attempted and successful in all We further correlated hMLH1 expression with the hMLH1 promoter tumors. Cases that had inadequate internal control tissue or inadequate methylation status of the MSI-H tumors. Eighteen UCSD MSI-H tumors tumor in the section or in which the staining was technically inadequate were included in this analysis. Of the MSI-H cases, 4 lacked expression of were eliminated. the hMSH2 protein. These 4 tumors were not included in this analysis, Statistical Analysis. Fisher’s exact test and the chi-square test were used because they were considered to belong to the HNPCC spectrum. Of 45 as appropriate to test the associations between hMLH1 and hMSH2 protein total MSI-H tumors, 14 were missing data on one or both of hMLH1 expression and MSI status and to test associations within the MSI-H sub- methylation and protein expression and 4 were negative on hMSH2. The 27 group. The analyses involving MSI status were performed on 173 CALGB remaining MSI-H tumors were included in this analysis. tumor samples. Analyses within the MSI-H subgroup included data from an Of 15 MSI-H cancers with loss of hMLH1 expression, 14 (93%) were additional 18 UCSD tumor samples. Sample sizes in each analysis varied also methylated while 42% MSI-H cancers (5/12) with positive immunos- according to the numbers of samples analysed for each of MSI status, taining for hMLH1 were methylated at the hMLH1 promoter (p = 0.009) hMLH1 and hMSH2 protein expression and hMLH1 methylation. All (Table 3). We thus were able to demonstrate the close relationship of statistical analyses were performed at the CALGB statistical center. hMLH1 protein expression with the hMLH1 promoter methylation status. Ability of MSI Markers to Determine the MSI Phenotype. Previous studies have reported a high accuracy of specific MSI markers in detecting RESULTS the MSI-H phenotype, especially for the highly monomorphic mononu- MSI Status, Correlation with hMLH1 Promoter Methylation and cleotide marker BAT26. We compared the sensitivity and specificity of MMR-Gene Expression. Of the 173 tumors from CALGB, 111 (64%) each individual marker of the Bethesda panel for its ability to define the were MSS, 35 (20%) were MSI-L and 27 (16%) were MSI-H (Table 1). In MSI-H (n = 45) phenotype based on all 5 markers. All 45 MSI-H tumors order to rule out further undetected MSI, we performed MSI analysis with (from CALGB and UCSD) were included in this analysis. We found that no additional mononucleotide (TGFβRII, IGFIIR, BAX, hMSH3, hMSH6), individual marker on its own was able to reliably detect the MSI-H pheno- di-nucleotide (D3S1029, D17S261, D18S64, D18S69 and D18S474) and type. Sensitivity of the individual MSI markers in detecting MSI-H was tetra-nucleotide markers (MYCL1). Even by adding these markers, the overall MSI pattern did not change. We further investigated the methylation status of the hMLH1 promoter. We were able to successfully perform the MSP assay in 20 MSI-H and 27 MSI-L cancers (Fig. 1). Thirty-five MSS tumors were non-randomly selected for MSP analysis of which 34 were successful. As expected, most of the MSS (28/34, 82%) and MSI-L (22/27, 81%) cancers were unmethylated at the hMLH1 promoter, while 6 of 20 (30%) MSI-H cancers were unmethylated (p<0.0001) (Fig. 2). Subsequently, we performed expression analysis of the MMR proteins hMLH1 and hMSH2 in the CALGB tumor cohort (Table 2, Fig. 3). Of 108 MSS and MSI-L cancers investigated, 107 (99%) expressed hMLH1. All but one tumor were unmethylated. Of the MSI-H cancers, 11/20 (55%) expressed hMLH1 Table 2 hMLH1 EXPRESSION IN ALL TUMORS INVESTIGATED MSS MSI-L MSI-H tumors (n = 128) (n = 84) (n = 24) (n = 20) Figure 2. Methylation status according to the degree of MSI. The hMLH1 99% 100% 55% promoter methylation was investigated by MSP in each tumor group. www.landesbioscience.com Cancer Biology & Therapy 75 © 2004 Landes Bioscience. Not for distribution. THE MSI PHENOTYPE OF COLORECTAL CANCER Table 3 CORRELATION OF MMR GENE EXPRESSION AND METHYLATION STATUS IN MSI-H CANCERS (INCLUDING CALGB AND UCSD CASES) Tumors Methylated Unmethylated Total Lack of hMLH1 14 (93%) 1 (7%) 15 expression hMLH1 expression 5 (42%) 7 (58%) 12 Total 19 (70%) 8 (30%) 27 this, an important issue still remains as to how the difference in sen- sitivity of immunohistochemistry in MSI-H cancers demonstrated in our and previous studies can be best explained. One issue is the Figure 3. hMLH1 expression. hMLH1 protein expression was investigated in definition of the MSI phenotype itself. It has been convincingly all tumor groups by IHC. shown that the MSI-H phenotype is correlated in most cases with aberrant methylation of the hMLH1 promoter which influences the highest for the dinucleotide markers D17S250 and D5S346 with 76% and transcriptional activity of the gene. We therefore investigated the 73%, respectively. Specificity was best for BAT26 and BAT25 with 99% and promoter methylation status of our entire tumor cohort with the 95%, respectively (Table 4). MSI status. We found that most MSS/MSI-L cancers had an unmethylated and, presumably, transcriptionally active hMLH1 DISCUSSION promoter. In 99% of these cases, the tumors were shown to express hMLH1 and hMSH2 proteins by immunohistochemistry. However, Molecular classification of colorectal cancer based on its molecu- lar features has important implications regarding prognosis and 18% of the MSS and 19% of the MSI-L cancers showed methyla- tion. Surprisingly, only 70% of the MSI-H cancers demonstrated might influence future treatment strategies. It has been shown that MSI-H sporadic colon cancers have in vitro resistance to common- promoter hypermethylation. Correlating the immunohistochemistry findings and the promoter methylation status in MSI-H cancers, we ly used chemotherapeutic drugs compared to cancers with the MSS and MSI-L phenotype. It remains to be determined if this is also found a strong association between negative immunohistochemistry and promoter methylation. However, the association between relevant in vivo. Up to now, only one retrospective study showed that 5-FU based chemotherapy might influence overall and disease hMLH1 expression and promoter methylation status was not absolute, as 7% of the MSI-H tumors lacking hMLH1 expression free survival negatively in patients with stage II and III CRCs. The MSI-H phenotype arises through the defects of the human were unmethylated and 42% of the hMLH1 expressing neoplasms were methylated. Although it would have been interesting to obtain MMR system, notably through transcriptional silencing of the hMLH1 gene by aberrant methylation of its promoter. Promoter data on DNA sequencing on those MSI-H cases that retained hMLH1 or hMSH2 expression in order to investigate if those methylation has been demonstrated to inhibit hMLH1 protein expression. So far, MSI-H cancers have been diagnosed by MSI tumors harbor somatic missense mutations that inactivate protein function. Unfortunately the amount of tumor DNA available did testing using a panel of consensus MSI markers. By definition, tumors with band shifts in 30–40% or more of the tested MSI mark- not allow us to perform such analysis for the current study. All MSI-H tumors in this study were defined using Bethesda ers are scored as MSI-H. However, no study has yet systematically compared the correlation of MSI-H sporadic colon cancers diagnosed criteria. Our findings raise two issues: 1) whether this definition cor- rectly categorizes all MSI tumors; and 2) whether all tumors with the by conventional MSI testing with the hMLH1 promoter methylation status and hMLH1 protein expression, respectively. It seems feasible MSI-H phenotype develop through the same molecular mecha- nisms. It has been uniformly assumed that hMLH1 promoter that searching for cancers with a silenced hMLH1 gene by promoter methylation could all be performed by simply employing the MSP methylation is responsible for transcriptional silencing and is related to the MSI-H phenotype. In our study, only 70% of the MSI-H technique instead of PCR testing with 5 standard MSI markers. With this strategy, screening for these cancers could be facilitated. cancers are hypermethylated and 45% of the MSI-H cancers expressed hMLH1. Some of these cases can be explained by defects In our study we assessed the benefits of MSI testing, hMLH1 promoter methylation analysis and hMLH1 protein expression for of other MMR genes such as hMSH2 (4 cases in this study). Presumably, these were not sporadic cancers but rather belonged to the determination of sporadic CRC with the MSI-H pheno- 23,29,30 type. First, we demonstrated the expected frequency of the MSI phenotype in sporadic colorectal cancer with 16% of all cancers Table 4 SENSITIVITY AND SPECIFICITY OF INDIVIDUAL MSI MARKER being MSI-H. Interestingly, only 45% of these cancers showed absence of hMLH1 expression. Earlier studies have estimated sensi- Sensitivity Specificity tivity between 75% and 100% for negative immunostaining and the MSI-H phenotype. However, in our study only 1 of 108 MSS/MSI-L D2S123 69% 85% cancers did not express hMLH1 protein suggesting a high specificity of D5S346 76% 79% MMR gene expression in non-MSI-H cancers. This is in accordance D17S250 73% 85% with others, all showing a specificity of 100% of immunohisto- BAT25 67% 95% chemistry in correctly diagnosing the MSS phenotype in cancers 22,23,29-31 BAT26 71% 99% with positive hMLH1 and hMSH2 expression. Despite 76 Cancer Biology & Therapy 2004; Vol. 3 Issue 1 © 2004 Landes Bioscience. Not for distribution. THE MSI PHENOTYPE OF COLORECTAL CANCER 8. Laiho P, Launonen V, Lahermo P, Esteller M, Guo M, Herman JG, et al. Low-level the familial tumor syndrome HNPCC. Our findings are in agree- microsatellite instability in most colorectal carcinomas. Cancer Res 2002; 62:1166-70. ment with a recent study showing that only 47% of MSI positive 9. Kane MF, Loda M, Gaida GM, Lipman J, Mishra R, Goldman H, et al. Methylation of the sporadic colorectal tumors with absent hMLH1 expression were also hMLH1 promoter correlates with lack of expression of hMLH1 in sporadic colon tumors and mismatch repair-defective human tumor cell lines. Cancer Res 1997; 57:808-11. hypermethylated at the hMLH1 promoter. It is not known what 10. Whitehall VL, Walsh MD, Young J, Leggett BA, Jass JR. Methylation of O-6-methylgua- role other MMR or DNA repair genes play in the development of nine DNA methyltransferase characterizes a subset of colorectal cancer with low-level DNA the MSI-H phenotype. So far, it has not been demonstrated that any microsatellite instability. Cancer Res 2001; 61:827-30. 11. Jass JR, Biden KG, Cummings MC, Simms LA, Walsh M, Schoch E, et al. Characterisation of the known MMR genes plays a major role in causing the MSI-H 6 of a subtype of colorectal cancer combining features of the suppressor and mild mutator phenotype in sporadic colorectal cancer. Only the O -MGMT gene, pathways. J Clin Pathol 1999; 52:455-60. when methylated and transcriptionally silenced, has been clearly 12. Chao A, Gilliland F, Willman C, Joste N, Chen IM, Stone N, et al. Patient and tumor char- acteristics of colon cancers with microsatellite instability: A population-based study. Cancer linked to cancers with the MSI-L, but not the MSI-H, phenotype. Epidemiol Biomarkers Prev 2000; 9:539-44. The question of how the MSI phenotype should be truly defined 13. Hawkins NJ, Ward RL. Sporadic colorectal cancers with microsatellite instability and their remains unanswered. We demonstrated that most cancers which did possible origin in hyperplastic polyps and serrated adenomas. J Natl Cancer Inst 2001; 93:1307-13. not show immunoreactivity for hMLH1 or hMSH2 were also 14. Jass JR, Iino H, Ruszkiewicz A, Painter D, Solomon MJ, Koorey DJ, et al. Neoplastic pro- methylated for hMLH1. Conversely, most of the hMLH1 and gression occurs through mutator pathways in hyperplastic polyposis of the colorectum. Gut hMSH2 expressing MSI-H cancers were unmethylated. MSI-H was 2000; 47:43-9. diagnosed using the reference panel of 5 MSI standard markers and 15. Ribic CM, Sargent DJ, Moore MJ, Thibodeau SN, French AJ, Goldberg RM, et al. Tumor microsatellite-instability status as a predictor of benefit from fluorouracil-based adjuvant no significant difference was evident between the instability pattern chemotherapy for colon cancer. N Engl J Med 2003; 349:247-57. of hMLH1 expressing and non-expressing cancers with respect to 16. Aebi S, Kurdi-Haidar B, Gordon R, Cenni B, Zheng H, Fink D, et al. Loss of DNA mis- the markers tested. match repair in acquired resistance to cisplatin. Cancer Res 1996; 56:3087-90. 17. Carethers JM, Chauhan DP, Fink D, Nebel S, Bresalier RS, Howell SB, et al. Mismatch repair All MSI markers used in the study had a high specificity but a proficiency and in vitro response to 5-fluorouracil. Gastroenterology 1999; 117:123-31. rather low sensitivity, suggesting that no marker on its own was able 18. Arnold CN, Goel A, Boland CR. Role of hMLH1 hypermethylation in drug resistance to to reliably detect all MSI-H cancers. However, when performing 5-fluorouracil in colorectal cancer cell lines. Int J Cancer 2003; 106:66-73. MSI analysis comparing MSI-H and hMLH1 hypermethylated or 19. Bocker T, Diermann J, Friedl W, Gebert J, Holinski-Feder E, Karner-Hanusch J, et al. Microsatellite instability analysis: A multicenter study for reliability and quality control. MSI-H and unmethylated cancers, all markers but the dinucleotide Cancer Res 1997; 57:4739-43. repeat D2S123 had a high detection rate up to 90% for this selection 20. Dietmaier W, Wallinger S, Bocker T, Kullmann F, Fishel R, Ruschoff J. Diagnostic microsatellite instability: Definition and correlation with mismatch repair protein expres- of cancers. MSI-H cancers might originate from different pathways, sion. Cancer Res 1997; 57:4749-56. one being caused by silencing of the hMLH1 gene and others by yet 21. Boland CR, Thibodeau SN, Hamilton SR, Sidransky D, Eshleman JR, Burt RW, et al. A unrecognized causes. national cancer institute workshop on microsatellite instability for cancer detection and The issue which should be further addressed in future studies is familial predisposition: Development of international criteria for the determination of microsatellite instability in colorectal cancer. Cancer Res 1998; 58:5248-57. the extent and amount of promoter methylation within the hMLH1 22. Chaves P, Cruz C, Lage P, Claro I, Cravo M, Leitao CN, et al. Immunohistochemical detec- promoter causing transcriptional silencing of the gene. Earlier studies tion of mismatch repair gene proteins as a useful tool for the identification of colorectal car- cinoma with the mutator phenotype. J Pathol 2000; 191:355-60. reported a significant concordance between methylation of a 5’ 23. Lindor NM, Burgart LJ, Leontovich O, Goldberg RM, Cunningham JM, Sargent DJ, et promoter region (also called region A) with the MSI-H phenotype. al. Immunohistochemistry versus microsatellite instability testing in phenotyping colorec- Others reported a further downstream area of the promoter (region C) tal tumors. J Clin Oncol 2002; 20:1043-8. being more closely related to MSI-H cancers. However, their study 24. Salahshor S, Koelble K, Rubio C, Lindblom A. Microsatellite instability and hMLH1 and hMSH2 expression analysis in familial and sporadic colorectal cancer. Lab Invest 2001; was performed in various colorectal tumor cell lines and may not be 81:535-41. directly extrapolated to the in vivo setting. One recent study exam- 25. Menigatti M, Di Gregorio C, Borghi F, Sala E, Scarselli A, Pedroni M, et al. Methylation ined the methylation status of the whole hMLH1 promoter and pattern of different regions of the MLH1 promoter and silencing of gene expression in hereditary and sporadic colorectal cancer. Genes Chromosomes Cancer 2001; 31:357-61. concluded that MSI-H cancers usually possess a full methylation 26. 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THE MSI PHENOTYPE OF COLORECTAL CANCER THE FOLLOWING INSTITUTIONS PARTICIPATED IN THE STUDY Institution Name Location PI Grant # CALGB Statistical Office Durham, NC Stephen George, Ph.D supported by CA33601 Dana Farber Canc78er Institute Boston, MA George P. Canellos, MD supported by CA32291 Dartmouth Medical School-Norris Cotton Lebanon, NH Marc Ernstoff MD supported by CA04326 Cncr Ctr Massachusetts General Hospital Boston, MA Michael L. Grossbard, M.D. supported by CA12449 Mount Sinai School of Medicine New York, NY Lewis Silverman, MD supported by CA04457 Rhode Island Hospital Providence, RI William Sikov, M.D. supported by CA08025 Roswell Park Cancer Institute Buffalo, NY Ellis Levine, M.D. supported by CA02599 Southeast Cancer Control Consortium Inc. Goldsboro, NC James N. Atkins, M.D. supported by CA45808 SUNY Upstate Medical University Syracuse, NY Stephen L. Graziano, M.D. supported by CA21060 The Ohio State University Columbus, OH Clara D. Bloomfield, MD supported by CA77658 University of California at San Diego San Diego, CA Stephen Seagren, MD supported by CA11789 University of California at San Francisco San Francisco, CA Alan Venook, MD supported by CA60138 University of Chicago Medical Center Chicago, IL Gini Fleming, M.D. supported by CA41287 University of Illinois at Chicago Chicago, IL David Gustin, M.D. supported by CA74811 University of Iowa Iowa City, IA Gerald Clamon, MD supported by CA47642 University of Maryland Cancer Center Baltimore, MD David Van Echo, M.D. supported by CA31983 University of Massachusetts Medical Center Worcester, MA Mary Ellen Taplin, M.D. supported by CA37135 University of Minnesota Minneapolis, MN Bruce A Peterson, M.D. supported by CA16450 University of Missouri/Ellis Fischel Cancer Columbia, MO Michael C Perry, M.D. supported by CA12046 Center University of North Carolina at Chapel Hill Chapel Hill, NC Thomas C. Shea, M.D. supported by CA47559 University of Tennessee Memphis Memphis, TN Harvey B. Niell, M.D. supported by CA47555 Wake Forest University School of Medicine Winston-Salem, NC David D. Hurd, MD supported by CA03927 Walter Reed Army Medical Center Washington, DC John C. Byrd, M.D. supported by CA26806 78 Cancer Biology & Therapy 2004; Vol. 3 Issue 1 © 2004 Landes Bioscience. Not for distribution.

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Published: Jan 1, 2004

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