Microsatellite Instability and MLH1 Promoter Methylation in Human Retinoblastoma

Kwong Wai Choy,1 Chi Pui Pang,1 Dorothy S. P. Fan,1 Thomas C. Lee,2 Jiang Hua Wang,1 David H. Abramson,2 Kwok Wai Lo,3 Ka Fai To,3 Christopher B. O. Yu,1 Katherine L. Beaverson,2 Kin Fai Cheung,1 and Dennis S. C. Lam1

PURPOSE. To investigate the link between microsatellite insta- he development of cancer is a multistep process involving bility and epigenetic silencing of the MLH1 gene in the human Tsomatic activation of proto-oncogenes, inactivation of tu- retinoblastoma genome. mor suppressor genes, and epigenetic alterations such as DNA 1,2 METHODS. Methylation at the 5Ј region of MLH1 was studied, methylation. Genetic and epigenetic alterations that have along with its protein expression level by using immunohisto- been determined in retinoblastoma include loss of heterozy- chemical staining in 51 retinoblastoma tumors and 2 retino- gosity (LOH) at the RB1 locus and hypermethylation of tumor blastoma cell lines. Also assessed was the genomic stability of suppressors including RB1, RASSF1A,3–6 and MGMT,7 a DNA 26 retinoblastoma DNAs from microdissected tumor tissue and repair gene. Another cardinal feature of cancer cells is genomic matched normal retina tissue obtained from the same patient instability. In human tumors, genetic alterations can be divided by microsatellite instability (MSI) analysis. The National Cancer into at least four major categories: subtle DNA sequence Institute–designed reference panel, and 85 markers on chro- changes, such as microsatellite instability (MSI); chromosomal mosomes 1, 6, 9, and 13 were used. instability; chromosomal translocation; and gene amplification RESULTS. Hypermethylation of the MLH1 promoter was de- or deletion. All except MSI have been described in retinoblas- tected in the WERI-Rb1 cell line and in 34 (67%) of the 51 toma. MSI may be a molecular marker of tumorigenesis, and tumors, but not in cell line Y79 and the other 17 tumors. MLH1 the MSI assay may be useful for assessment of genetic aberra- hypermethylation was associated with null MLH1 protein ex- tions in specific human tumor types and stages. Analysis of pression (P Ͻ 0.0005) and with well-differentiated histology microsatellite markers at a specific locus not only helps to (P Ͻ 0.05). MSI at three markers (D2S123, D6S470, and identify tumor-related genes, but also contributes to prognostic D13S265) was frequently identified among 26 retinoblastoma indications.3,8–9 specimens with matched normal DNA. Among these 26 reti- MSI is a genome-wide alteration characterized by a global noblastomas, high-frequency MSI (MSI-H) tumors were de- instability phenomenon affecting repetitive microsatellite se- tected in 19% (5/26) and low-frequency MSI (MSI-L) in another quences. First discovered in colorectal cancers and subse- 19% (5/26). The remaining 62% (15/26) were genetically stable quently in many other cancers,10,11 it is caused by a failure of (MSS). MSI status (MSS, MSI-L, and MSI-H) was not associated ϭ the DNA replication error repair system to repair errors during with MLH1 promoter hypermethylation (P 0.088; Kruskal- 12,13 Wallis test). DNA replication. The extent of MSI varies considerably in different tumors. In MSI, there is frequent accelerated accumu- CONCLUSIONS. Epigenetic silencing of the DNA repair gene lation of single-nucleotide alterations in the length of repetitive MLH1 by promoter hypermethylation is a frequent event in microsatellite sequences that occurs ubiquitously throughout retinoblastoma. The results showed that somatic genetic the genome. Tumors displaying MSI occur as a result of germ- changes involving MSI occur in a subset of retinoblastoma and line mutations in certain genes, such as MLH1, MSH2, MSH6, implicated the presence of a defective DNA mismatch repair MLH3 pathway resulting in MSI in retinoblastoma. (Invest Ophthal- and , that compose the DNA mismatch repair system, mol Vis Sci. 2004;45:3404–3409) DOI:10.1167/iovs.03-1273 whereas in sporadic cancers, methylation of the MLH1 promoter appears to be the dominant mechanism leading to MSI.14,15 MSI has also been implicated in tumor development and clinical prognosis.16 From the Departments of 1Ophthalmology and Visual Sciences In retinoblastoma, inactivation of RB1 occurs in both famil- and 3Anatomical and Cellular Pathology, The Chinese University of 2 ial and sporadic cases. Heritable susceptibility to retinoblas- Hong Kong, Hong Kong, China; and the New York Presbyterian toma is transmitted in the autosomal-dominant mode, with 80% Hospital, Weill Medical College, Cornell University, New York, New 17 York. to 90% penetrance. In vitro studies and epigenetic investiga- Supported in part by Grant CUHK4091/01M from the Research tions have shown that RB1 gene alteration is a prerequisite for Grants Council of the Hong Kong Special Administrative Region, tumorigenesis.7,18 However, the presence of genomic instabil- China. ity including MSI and chromosome instability (CIN) in the Submitted for publication November 21, 2003; revised March 25, retinoblastoma genome19,20 indicate that such genetic instabil- 2004; accepted April 13, 2004. ity may have the putative oncogenic effects necessary for Disclosure: K.W. Choy, None; C.P. Pang, None; D.S.P. Fan, None; T.C. Lee, None; J.H. Wang, None; D.H. Abramson, None; retinoblastoma formation. We sought to determine whether K.W. Lo, None; K.F. To, None; C.B.O. Yu, None; K.L. Beaverson, MSI in tumor cells is associated with development of retino- None; K.F. Cheung, None; D.S.C. Lam, None blastoma and to explore the link between MSI and MLH1 gene The publication costs of this article were defrayed in part by page methylation in retinoblastoma. Methylation status of the CpG charge payment. This article must therefore be marked “advertise- island of the MLH1 promoter on retinoblastoma tissues was ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. studied. We performed microsatellite analysis using the 10 Corresponding author: Chi Pui Pang, Department of Ophthalmol- ogy and Visual Sciences, The Chinese University of Hong Kong, Hong reference panel markers of the National Cancer Institute (NCI), Kong Eye Hospital, 147K Argyle Street, Kowloon, Hong Kong; together with 85 markers on chromosomes 1, 6, 9, and 13, [email protected]. where prevalent abnormalities had been reported.8–9,20

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PCR using primers specific for methylation or nonmethylation sequences discriminated methylated from nonmethylated DNA. The PCR program: 95°C for 12 minutes followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 30 seconds, and a final extension for 7 minutes at 72°C. Microsatellite DNA Markers and MSI Analysis DNA extracted from the microdissected tumor cells and matched normal DNA from the same person were used for MSI analysis using fluorescence-labeled primers on 10 microsatellite markers recommended by NCI (D2S123, D5S346, D7S501, D10S197, D17S250, D18S58, BAT25, BAT26, BAT40, and D3S1611) to distinguish the form of genomic instability in the RB genome.22 We also analyzed 85 polymorphic microsatellite markers on chromosomes 1, 6, 9, and 13, with an average spacing of 10 cM and heterozygosity of 0.79. These were fluorescence labeled (Prism Linkage Mapping Set-MD10; Applied Biosystems, Inc. [ABI], Foster City, CA). All 95 markers were selected from the Geńe´thon linkage map on the basis of chromosomal location and heterozygosity.23 PCR products were electrophoresed on a se- quencer (377 Prism; ABI), and fluorescent signals from the different sized alleles were analyzed (Genotyper ver. 2.1 and Genescan, ver. 3.1 software; ABI). A given informative marker was considered to display MSI, if one or both alleles in the tumor DNA exhibited size variation FIGURE 1. MSP analysis of the MLH1 gene in retinoblastoma tissues. due to expansion or contraction of the repeat sequences in compari- This is a typical example of MSP analysis in retinoblastoma samples of R4, R9, R15, R40, and R38, which are microsatellite unstable, and of son with matched normal DNA from the same individual (Fig. 2). R2, R13, and R50, which are microsatellite stable. The PCR products in Tumors with high-frequency MSI (MSI-H) have instability in three or lane M indicate the presence of methylated alleles and in lane U of more markers, whereas tumors with low-frequency MSI (MSI-L) have nonmethylated alleles. In vitro SssI methyltransferase-treated and un- instability in less than three markers.22 Tumor specimens showing no treated DNA from normal lymphocytes were used as the positive apparent instability were designated microsatellite stable (MSS).22 MSI control for methylation and nonmethylation, respectively. was interpreted by computer (Genotyper; ABI). Immunohistochemistry MATERIALS AND METHODS Immunohistochemical staining on 5-␮m-thick formalin-fixed, paraffin- embedded tumor tissue was performed as described previously.7 In Specimen Preparation We retrieved 51 unrelated formalin-fixed, paraffin-embedded retinoblastoma specimens archived in the retinoblastoma clinic between 1990 and 2002 at the Hong Kong Eye Hospital (n ϭ 23) and New York Presbyterian Hospital (n ϭ 28). Informed consent and institutional review board approval was obtained from the participating institu- tions, and the protocol adhered to the provisions of the Declaration of Helsinki.

Laser Captured Microdissection We obtained more than 90% tumor cells from the retinoblastoma tissue samples by using a laser capture microdissection (LCM) system (PALM, Bernried, Germany) to select cancerous tissue cells on slides, according to the manufacturers’ protocol. Brieﬂy, the stained and dehydrated tissue section was overlaid with a thermoplastic ﬁlm mounted on an optically transparent cap. The visually selected areas of tumor cells were bound to the membrane by short, low-energy laser pulses, resulting in focal melting of the polymer. On average, approximately 20,000 tumor cells were yielded by LCM shots. They were incubated in 100 ␮L buffer containing 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 400 ␮g/mL proteinase K, and 1% Tween 20 for digestion at 55°C overnight. DNA was extracted with a DNA extraction kit (Qiagen, Hilden, Germany). Genomic DNA was also extracted from the corresponding normal eye tissue cells.

Methylation-Specific PCR Assay The methylation status in the CpG island 5Ј to the transcription start site of the MLH1 gene was examined by methylation-specific PCR FIGURE 2. MSI and LOH analyses in paired normal (N, top) and tumor 21 - (MSP) analysis, based on the sequence differences between methyl (T, bottom) tissue obtained by microdissection of a tissue specimen ated and nonmethylated DNA after bisulfite modification (CpGenome from the same individual. Electrophoregram of the dinucleotide repeat DNA Modification kit; Intergen, Purchase, NY). Nonmethylated cyto- marker D2S123 from a homozygous individual. Appearance of extra sine was converted to uracil, which is recognized as thymine by Taq alleles at lower molecular weights in tumor sample R4 indicates the polymerase. Methylated cytosine was not affected (Fig. 1). Subsequent presence of genomic instability (MSI).

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FIGURE 3. Immunohistochemical staining of MLH1 protein expression in typical human retinoblastoma sections. (A) Antibody reveals positive nuclear staining (brown) of MLH1 in normal human retinal cells, particularly in the retinal ganglion cell layer, INL, and ONL. The sections were counterstained with hematoxylin. (B) MSS tumor cells without MLH1 promoter methylation showing positive staining for MLH1 with intensity above normal adjacent retina cells. (C) Retinoblastoma cells stained positive in the tumor field with hetero- geneous MLH1 protein expression. (D) Retinoblastoma sections from patient R15 with methylation of the MLH1 promoter showing negativity for the MLH1 protein. (E) A nonmethylated Y79 cell line showing positive nuclear staining for MLH1. (A–D) Formalin fixed, paraffin-embedded tissue sections; (E) sections prepared by cytospin and fixed with 4% paraformaldehyde. Original mag- nification, ϫ400.

brief, the epitope site was retrieved by heating slides twice for 5 Statistics Analysis minutes in a microwave. After the endogenous peroxidase was ␹2 blocked with 0.6% hydrogen peroxide, the tissue sections were incu- The test, Kruskal-Wallis test, and Kaplan-Meier curves were used to bated overnight at 4°C with mouse monoclonal antibody MLH1 (BD- analyze the MSI in relationship to the methylation status of the MLH1 Ͻ PharMingen, San Diego, CA) at a 1:100 dilution containing 5% normal gene or the expression of the gene to any clinical significance. P sheep serum. Sections were rinsed with PBS (0.1 M phosphate buffer 0.05 was considered statistically significant. NaCl), incubated with biotinylated anti-mouse IgG (Dako, Carpinteria, CA) according to the instructions in the avidin-biotin complex staining ESULTS kit (Vectastain Elite ABC kit; Vector Laboratories, Burlingame, CA), and R finally counterstained with hematoxylin. Immunoreactive cells were MLH1 Promoter Methylation and Expression identified by their brown nuclei, whereas negative-staining cells had blue nuclei from hematoxylin counterstaining. Negative controls were Our MSP analysis (Fig. 1) detected hypermethylation of the generated by omitting the primary antibody in each case. Two of the MLH1 promoter in 34 (67%) of the 51 tumors and hemimethy- authors (KWC, KFC) examined the entire tumor area under the micro- lation in the WERI-Rb1 cell line. No MLH1 gene promoter scope for consensus (at least five ϫ100 fields). We scored tumors methylation was detected in the Y79 retinoblastoma cell line or positive for MLH1 protein expression when immunohistochemical the remaining 17 (33%) of the 51 retinoblastoma specimens. staining revealed the presence of MLH1 protein in more than 80% of Immunohistochemical staining for MLH1 protein expression the cells in the tumors investigated: (ϩϩ) More than 80% of the tumor showed that in normal retina, MLH1 was predominately cells were positive and the staining intensity was the same or above present in the nuclei of retinal ganglion cells and inner nuclear normal adjacent retina cells; (ϩ) positive staining in two thirds of the layer (INL). Fainter nuclear expression was found in some cells tumor field and cells with equal intensity compared with normal of the outer nuclear layer (ONL; Fig. 3A). In retinoblastoma, the adjacent cells; and (Ϫ) tumor cells are completely negative for immu- intensity of nuclear MLH1 immunostaining varied from posi- nohistochemical stain. tive, reduced to negative (Figs. 3B–D). For the 15 nonmethyl-

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TABLE 1. Clinical and Pathologic Features of the 26 Retinoblastoma Patients with Matched Normal and Cancerous Retinal Tissue Cells in MSI Analysis

Age at Eye Patient Diagnosis Survival MSI MLH1 MLH1 Optic Nerve Number Sex (y) (mo) Status Methylation Protein Invasion Recurrence Laterality Histology Treatment

R40 M Ͻ1 2 MSI-L ϪϩϪ NR Bi Di Ϫ R38 M 2 24 MSI-L Ϫϩϩ NR Bi Di ϩ R2 M 1 11 MSS ϪϩϩϪ NR Bi Uni Ϫ R13 F 2 24 MSS ϪϩϪ R Bi Uni ϩ R44 F Ͻ1 4 MSI-H ϩϪϪ NR Bi Di Ϫ R45 F 2 24 MSS ϩϪϩ NR Bi Di ϩ R47 M 2 24 MSS ϩϩϩϪ NR Bi Uni ϩ R48 M 1 12 MSS ϪϩϩϪ NR Ui Uni Ϫ R49 M Ͻ1 2 MSS ϪϩϩϪ NR Bi Uni Ϫ R50 M 1.5 11 MSS ϪϩϪ NR Bi Uni Ϫ R4 M 3 32 MSI-H ϩϩϪ NR Ui Uni ϩ R9 M Ͻ1 3 MSI-H ϩϪϪ NR Bi Di ϩ R10 M 2 27 MSS ϪϩϩϪ NR Ui Uni ϩ R15 F 1.5 17 MSI-H ϩϪϪ NR Bi Uni Ϫ Yu62 M 2.5 12 MSS ϪϩϪ NR Ui Di Ϫ Yu63 M 2 23 MSS ϩϩϪ NR Bi Uni Ϫ Yu64 M Ͻ1 4 MSS ϪϩϪ NR Bi Uni Ϫ Yu65 M 3 36 MSI-H ϪϩϪ NR Bi Uni ϩ Yu66 M Ͻ1 4 MSS ϩϪϪ NR Bi Di Ϫ Yu67 M 2 23 MSS ϪϩϩϪ NR Ui Di ϩ Yu68 F 1 12 MSI-L ϪϩϪ RUiDiϩ Yu69 F Ͻ1 7 MSI-L ϩϪϪ NR Ui Di ϩ Yu70 M Ͻ1 1 MSI-L ϪϩϪ NR Bi Di Ϫ Yu71 M Ͻ1 3 MSS ϪϩϩϪ NR Bi Di Ϫ R54 F 2 24 MSS ϪϩϪ NR Ui Uni ϩ Yu72 M 3 5 MSS ϪϩϩϪ NR Bi Uni ϩ

Null MLH1 protein expression was associated with MLH1 promoter hypermethylation (P Ͻ 0.0005). Treatment refers to prenucleation treatment with iodine plaque or chemo- or cyrotherapy. Scoring system for MLH1 protein detection is described in the Methods section. Bi, bilateral; Ui, unilateral; Di, well-differentiated; uni, poorly differentiated. For recurrence: R, local tumor recurrence; NR, no recurrence.

ated samples including the Y79 cell line (Figs. 3B, 3E), there (Ͼ40%) among the MSI-H cases. Hypermethylation at the was more prominent expression in differentiated tumor cells MLH1 promoter was found in 4 of 16 MSS specimens (25%; but with less intensity in the undifferentiated cells. No MLH1 Clopper–Pearson 95% confidence interval [CI]: 7.3%–52.4%), 1 protein was detected in 29 of the 34 retinoblastoma specimens of 5 MSI-L (20%; Clopper–Pearson 95% CI: 0.5%–71.6%), and 4 with MLH1 hypermethylation, and the rest (5/34) showed of 5 MSI-H (80%; Clopper–Pearson 95% CI: 28.43%–99.5%). MSI presence of focally positive cells and impaired MLH1 expres- status (MSS, MSI-L, and MSI-H) was not associated with MLH1 sion (data not shown). Null MLH1 protein expression was promoter hypermethylation (P ϭ 0.088; Kruskal-Wallis test). significantly associated with hypermethylation of the MLH1 promoter (P Ͻ 0.0005; Table 1). Association between MSI and Clinical Features Microsatellite Instability There was no significant association between MSI status and the clinical and pathologic features of retinoblastoma in age of From the 51 retinoblastoma specimens, normal tissue cells diagnosis, histologic differentiation, optic nerve invasion be- were successfully dissected and removed from cancerous cells. yond the lamina cribrosa, and treatment before enucleation Genomic DNA was extracted separately from the cancer cells (Table 1). Two of the 26 patients had optic nerve invasion and and normal cells for MSI analysis. A sufficient amount of another two had a recurrence, all in MSS or MSI-L tumors genomic DNA was obtained from cancer cells of all 51 speci- (Table 1). Although none of the MSI-H patients had a recur- mens but from normal cells in only 26 specimens. MSI was rence or optic nerve invasion, the number of samples in this studied in these 26 pairs of specimens with DNA from matched study is too small to suggest MSI status to be of prognostic cancer and normal retina tissue cells from the same patient value. (Table 1). DNA band patterns of tumor and normal cell pairs were compared. MSI was detected in tumors at both differen- Association between Methylation Status and tiated and undifferentiated stages of malignancy but not in Clinical Features constitutional normal DNA. MSI-H was found in 19% (5/26) of the tumors, which exhibited instability at three or more mark- The 51 retinoblastoma cases in this study had different clinical ers with size shifts. MSI-L was detected in another 19% (5/26) and pathologic features (Table 2). All patients were diagnosed of the tumors, with instability at less than three markers (Table under the age of 5 years and followed up (minimum 2 months, 1). The remaining 62% (16/26) were regarded as microsatellite median 32 months, maximum 8 years). All were still surviving stable (MSS), according to the NCI.22 A high level of instability at the time of this report except one patient (R2), who had was observed in the five MSI-H tumors. In total, nine markers osteosacroma develop in the right maxilla and died at 12 years showed MSI: D1S450, D1S199, D1S252, D6S470, D6S1581, of age. There was no association between MLH1 promoter D9S285, D9S161, D13S265, and D2S123. Three of them, methylation and all the clinical or pathologic features, except D6S470, D2S123, and D13S265, were frequently identified that it was significantly associated with retinoblastoma with

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TABLE 2. MLH1 Methylation Status of All 51 Tumor Specimens and samples is required. In addition, two of the four MSI-H retino- Their Clinical and Pathologic Features blastoma specimens showed MSI in the D2S123 locus, which is located within the MSH2 gene (Fig. 2). In contrast to the MLH1 Methylation Status other retinoblastoma specimens, there was no MSH2 protein expression in these two cases (data not shown). Therefore, we Methylated Nonmethylated suggest that the MSI observed in these two retinoblastoma cases may also be associated with lesion in the MSH2 gene, as Cases (n)3417 25 Laterality (n) has been reported in colorectal carcinoma. Unilateral 14 6 MSI varies considerably in different tumors and the degree Bilateral 20 11 of MSI reflects the frequency of strand slippage events during 12 Histological classification (n)* replication and the efficiency of subsequent mismatch repair. Differentiated 27 8 In colorectal cancer MSI was detected on 5q, 17p, and 18q and Undifferentiated 7 9 was associated with increased patient survival and tumor loca- Eye survival tion in the colon.16 In this study, the absence of local tumor Median (mo) 17 12 recurrence and optic nerve invasion among MSI-H patients may Optic nerve involvement (n)1 2indicate survival advantages among the MSI-H retinoblastoma Tumor recurrence (n)32 patients, as has been documented in patients with hereditary Primary nonocular cancer (n) 14,16 Presence 2 1 colorectal cancer of the MSI-H genotype. However, the Absence 32 16 number of cases in this study was too few to draw a statistical Pre-enucleation treatment (n) correlation between MSI and clinical features. Meanwhile, us- With iodine plaque or chemo- or ing a panel of 85 microsatellite markers on chromosomes 1, 6, cryotherapy 15 8 9, and 13, together with the 10 markers recommended by the No treatment 19 9 NCI for MSI analysis, we frequently found the presence of MSI Family history (n) in 3 markers (D2S123, D6S470, and D13S265), particularly With family history 3 1 among our MSI-H retinoblastoma cases. Only D2S123 was Sporadic 31 16 commonly identified in other cancers, such as colorectal14,16 28 * Statistically significant; P Ͻ 0.05. and gastric cancer. Our results suggest that a different panel of microsatellite markers should be used to assess MSI in retinoblastoma. It is notable that van der Wal et al.20 detected well-differentiated histology (P Ͻ 0.05; Table 2). Although the no MSI in retinoblastoma by using the five reference panel median eye survival of patients with methylation was shorter markers from the NCI. than that of those without methylation, 12 against 17 months Genomic instability at the retinoblastoma genome is mostly for patients with and without methylation, respectively, the reported at the nucleotide level, such as base substitutions, difference was not statistically significant, based on the Kaplan- small deletions or insertions in the Rb1 gene. In contrast, Meier curves. somatic instability is observed either as a substantial change in repeat length or LOH at particular loci. We found that impaired DISCUSSION expression of MGMT was associated with promoter methylation in retinoblastoma7 and the presence of genome instability Human retinoblastoma contains genomic alterations. Some of in retinoblastoma.19 In this study, the identification of epige- these genetic defects may contribute to the development and netic silencing of MLH1 in a high proportion of retinoblastoma progression of retinoblastoma or even resistance to therapy. In cases (67%, 34/51) shows MLH1 promoter hypermethylation this study, we investigated MLH1 gene hypermethylation, to be a frequent event in retinoblastoma, providing additional which implicates a defect in mismatch repair and MSI in the evidence of involvement of a DNA repair defect in the devel- retinoblastoma genome and their possible links to tumorigen- opment of retinoblastoma. There is also an association be- esis. We detected MSI-H at a moderate frequency (19%) in the tween MLH1 hypermethylation and impairment of MLH1 pro- 26 available cases (Table 1). This was much lower than MSI-H tein production, as is known in gastric cancer.27 The presence in hereditary nonpolyposis colorectal cancer (75%Ϫ100%)15 of MLH1 methylation, irrespective of laterality and treatments but was comparable to ovarian cancer (17%)24 and sporadic indicating that such epigenetic silencing was due neither to colorectal carcinoma (15%).15 In human cancers, particularly chemotherapy nor to cryotherapy before enucleation nor was hereditary nonpolyposis colorectal cancer, most of the MSI it related to the hereditary or sporadic nature of the disease. was associated with genetic mutations or epigenetic alterations The epigenetic silencing of the MLH1 gene could be an early in the mismatch repair genes MSH2 and MLH1, located on event that occurs during tumor development. In addition, we 2p22-p21 and 3p21.3, respectively.13,15 Most tumors with MSI identified the presence of MSI in a subset of retinoblastoma lack MSH2 and MLH1 expression, indicating genetic alter- samples. The findings in this and our previous study7 indicated ations in these mismatch repair genes13,14,25 to be associated that epigenetic silencing of DNA repair genes MLH1 and with hypermethylation of the MLH1 promoter.22 MGMT may play a role in the development of retinoblastoma. We found CpG methylation of the MLH1 promoter in retinoblastoma samples with different MSI status (Table 1). Al- Acknowledgments though retinoblastoma samples with MSI-H had higher frequency than the MSI-L and MSS samples, statistical comparison The authors thank Nongnart Chan and Winnie Li for support and is not meaningful due to small sample size. 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