ASSESSMENT OF MLH1 PROMOTER METHYLATION IN ENDOMETRIAL CANCER USING PYROSEQUENCING TECHNOLOGY OBJECTIVES

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1 ASSESSMENT OF MLH1 PROMOTER METHYLATION IN ENDOMETRIAL CANCER USING PYROSEQUENCING TECHNOLOGY Authors: Duilio Della Libera, Alessandra D Urso, Federica Modesti, Georgeta Florea, Marta Gobbato Hospital: Ospedale S.Maria del Prato, ULSS 2 Feltre ( Bl ) OBJECTIVES Among the processes that control cell proliferation, DNA methylation represents an important epigenetic mechanism for the regulation of gene expression and its deregulation is often associated with cancer development (Robertson KD, 2005). MLH1 is a mismatch repair (MMR) gene and represents one of the most studied genes for understanding the role of promoter methylation status in cancer (Capel et al., 2007). MLH1 promoter methylation is directly involved in tumour initiation and progression, and its methylation makes cells unable to repair DNA damage (Kane et al., 1997). DNA replication errors occur mainly at the level of microsatellite repeat sequences and give rise to the microsatellite instability (MSI) phenotype (Boland et l., 1998). MSI tumours may be sporadic or hereditary, as happens in patients affected by Lynch syndrome (LS). The main difference between sporadic and hereditary MSI is the cause of the MMR system defect. Hereditary MSI tumours are caused by a germline mutation of one of the MMR genes (usually MSH2 or MLH1, although mutation of MSH6 and PMS2 also occurs at a low frequency). Sporadic MSI tumours are caused by acquisition of somatic MLH1 promoter hypermethylation which often occurs in the context of global hypermethylation of gene promoters and is known as the CpG island methylator phenotype (Samowitz et al., 2005).

2 Given the evidences above, analysis of MLH1 promoter methylation status has proven useful as a marker in a screening algorithm for the classification of human neoplasias and the final diagnosis of LS. The published literature suggests that assessing the BRAF V600E mutation is highly associated with MLH1 promoter methylation in colorectal cancer (CRC) (Bessa et al., 2008). By contrast, MLH1 methylation analysis is required for the correct characterization of LSrelated endometrial carcinomas (ECs) because BRAF mutations have not been detected in these tumours (Kawaguchi et al., 2009; Kitayama et al., 2011). The aim of the present work was to develop and validate an MLH1 methylation assay suitable for characterizing both CRC and EC to complete the screening algorithm for LS as used in our laboratory, which is based on immunohistochemistry (IHC) of MMR proteins, MSI analysis and BRAF V600E mutation detection. METHODS DNA from CRC and EC samples were extracted using a QIAamp DNA FFPE Tissue Kit (Qiagen) by microdissecting 5 μmthick tissue sections. All tumours were analysed for MSI using a fivemononucleotide marker panel represented by NR21, NR22, NR24, BAT25 and BAT26. The amplified tumour DNA and the matching normal DNA derived from blood were analysed using capillary electrophoresis and GeneMapper software. Immunohistochemical staining of MMR proteins was performed on formalinfixed, paraffinembedded tissue. The primary antibodies were antihuman MutL protein homolog 1 (clone ES05), antihuman postmeiotic segregation increased 2 (clone EP51), antihuman MutS protein homolog 2 (clone FE11) and antihuman MutS protein homolog 6 (clone EP49) (all from Dako). Positive and negative controls were considered. CRC samples were subjected to BRAF mutation analysis using antiegfr MoAb response (BRAF status) (Diatech Pharmacogenetics). MLH1 promoter methylation was assessed using pyrosequencing technology on DNA extracted from 5 μmthick sections of CRC and EC tissue. DNA extracted was treated with sodium bisulfite for the conversion of unmethylated cytosine. Subsequently, the promoter regions between positions 248 and 178 from the transcription start site (TSS) and containing 8 CpG sites were amplified by realtime PCR using a specific primer set developed by PyroMark Assay Design software.

3 RESULTS Methylation assay setup. MLH1 core promoter methylation analysis was developed and applied to DNA extracted from CRC samples that might act as positive and negative controls. The former is represented by CRC samples characterized by MLH1/PMS2 protein loss, MSIH phenotype and BRAF V600E mutation, which are highly associated with MLH1 promoter hypermethylation, as mentioned above. The latter is represented by CRC samples characterized by retention of MLH1/PMS protein expression, MSIS phenotype and absence of the BRAF mutation. Sodium bisulfitetreated DNA was amplified by realtime PCR using a primer set specifically designed to amplify the region between positions 248 and 178 from the TSS and containing 8 CpG sites. Figure 1 : MLH1 methylation assay primers set Figure 2: MLH1 methylation assay. Amplification Plot and Melting Curve analysis of Negative Control samples (panels A1, A2, A3) and Positive Control samples (panels B1, B2, B3) Agarose gel electrophoresis of the obtained amplicons showed a 186 pb band, as expected. Melting curve analysis showed a single peak at Tm 76 ± 1 C for the negative control samples (Figure 2, panel A2 and A3) and a double peak at Tm 76 ± 1 C and 79 ± 1 C for the positive control samples (Figure 2, panel B2 and B3). This double peak was probably caused by the presence of two populations of DNA molecules, one derived from the tumour cells showing a pattern of hypermethylation (Tm 79 ± 1 C) and the other derived from normal stromal cell surrounding the neoplasia not characterized by methylation events (Tm 76 ± 1 C). A notemplate control (NTC) showed a single peak at Tm 73 C, which did not overlap with the diagnostic peaks and was probably caused by primer dimers formation (Figure 2, panel A2 and A3, B2, B3). The real methylation status of the MLH1 promoter was assessed by pyrosequencing analysis of the amplified sample by PyroMark Q96ID and PyroMark CpG software. Positive control samples showed a mean methylation greater than 80%. For negative control samples, the modulation percentage of each of the eight CpG sites ranged from a minimum of 0% to a maximum of 10%, with an average modulation percentage of 4%. This allowed us to establish a mean cutoff of 5% methylation to discriminate unmethylated samples from hypermethylated samples. Figure 3: Pyrosequencing analysis of positive control sample for MLH1 metylation

4 ICH and Molecular characterization of endometrial cancer samples. A series of 46 ECs were analysed for MMR protein expression by IHC. Thirtyeight of the analysed samples showed physiological expression of MLH1, PMS2, MSH2 and MSH6, whereas seven samples were characterized by loss of expression of one or two MMR members. In particular, five cases showed MLH1/PMS2 dimer loss, one case showed MSH2/MSH6 dimer loss and one case showed isolated loss of PMS2. Case number MLH1 PMS2 MSH2 MSH Focal expression Figure 4: ICH analisys of MMR protein expression; Normal endometrial tissue expression of MLH1 (A), PMS2 (C), MSH2 (E), MSH6 (G); endometrial tumor loss of expression of MLH1 (B), PMS2 (D), MSH2 (F), MSH6 (H) Table 1 : EC samples showing abnormal expression of MMR proteins. All 46 cases were subjected to MSI analysis using a panel of five quasimonomorphic mononucleotide markers (NR21, NR22, NR24, BAT25 and BAT26) which appeared to be more sensitive and specific than the NCI panel in determining the MSI status (Suraweera et al., 2002; Buhard et al., 2004). Figure 5 :Example of MSIAnalysis on Endometrial Cancer (EC) tissue samples. A: MSIS EC sample, all five markers are characterized by reference lenght; B: MSIH EC sample, all five markers show a deviation from reference lenght CASO MSI Among 46 cases analysed, 39 were stable and seven were MSIH (high level of instability) and showed an allelic shift of at least two markers. The correlation between the IHC and MSI analysis was 100%. 17 MSIH 24 MSIH 26 MSIH 35 MSIH 42 MSIH 43 MSIH 45 MSIH NR21 BAT26 BAT25 NR24 NR22 Table 2: MSIAnalysis, EC samples characterized by MSIH and molecular marker affected.

5 Validation of methylation assay in endometrial cancer samples The five MSHIH samples with MLH1/PMS protein loss were assessed for MLH1 promoter methylation status, together with five MSIS samples with MLH1/PMS2 protein retention as negative controls. All five MSIH endometrial tumours showed a pattern of hypermethylation with a mean methylation of 66% to 88%, whereas the five MSIS EC tumours were negative and had a mean methylation of 3% to 5%. Our results suggest that all five MSIH EC tumours with MLH1 protein loss could be considered to be of sporadic origin and exit from the screening algorithm for LS diagnosis. Figure 6 : MLH1 core promoter methylation analisys; example of hypermethylated MSIH EC sample Figure 7 : MLH1 core promoter methylation analisys; example of nonmethylated MSIS EC sample

6 DISCUSSION AND CONCLUSION MLH1 promoter methylation is a frequent event in EC, as described previously (Kanaya et al. 2003,Varley et al., 2009). Analysis of MLH1 promoter hypermethylation is fundamental during the molecular characterization of LSrelated EC because no other biological marker, such as the BRAF V600E mutation that is useful in CRC characterization, seems to be associated with MLH1 epigenetic silencing in endometrial tissue In 1999, Deng et al correlated the hypermethylation of a small proximal promoter region between nucleotides 248 and 178 from the TSS, defined as the C region, with the absence of MLH1 expression in a CRC cell line. In 2007, Capel et al confirmed and extended the results of Deng et al by analysing a series of 34 MSS and 38 MSI primary colorectal tumours. Their results underlined the importance of analysing the proximal but not the distal region of the MLH1 promoter when evaluating the hypermethylation events causing protein loss. Data obtained Kenya et al in 2003 established that hypermethylation of both the proximal and distal regions of the MLH1 promoter are found frequently in EC. These data support the notion that the MLH1 methylation profile might be tumourtype specific and that the degree, rather than the regional specificity, of methylation is responsible for the transcriptional silencing and decreased expression of MLH1 protein in EC. We decided to develop a single MLH1 methylation assay that would be suitable for both CRC and EC. We used pyrosequencing technology to analyse the proximal core promoter region, which is critical to the first type of neoplasia. Our analysis of the 8 CpG site between positions 248 and 178 showed that this sequence seems to be adequate for defining the hypermethylation status of MLH1 in EC. Our MLH1 methylation assay based on pyrosequencing technology proved to be suitable for the analysis of DNA samples derived from formalinfixed paraffinembedded specimens, which are normally considered difficult to analyse because of DNA degradation caused by the fixation process. Our method provides a sensitive and accurate prescreening test for discriminating sporadic tumours from hereditary forms of neoplasia. REFERENCES Bessa X, Balleste B, Andreu M et al. A prospective, multicenter, populationbased study of BRAF mutational analysis for Lynch syndrome screening. Clin. Gastroenterol. Hepatol. 2008; 6; Buhard,O., Suraweera,N., Lectard,A., Duval,A. and Hamelin,R. (2004) Quasimonomorphic mononucleotide repeats for high level microsatellite instability analysis. Dis Markers, 20, Boland CR, Thibodeau SN, Hamilton SR, Sidransky D, Eshleman JR, Burt RWet al. (1998). A National Cancer Institute workshop on microsatellite instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer. Cancer Res 58: Capel E., Flejou JF., Hamelin R. Assessment of MLH1 promoter methylation in relation to gene expression requires specific analysis. Oncogene (Short Communication), : p Kane MF, Loda M, Gaida GM, Lipman J, Mishra R, Goldman H et al. (1997). Methylation of the hmlh1 promoter correlates with lack of expression of hmlh1 in sporadic colon tumors and mismatch repairdefective human tumor cell lines. Cancer Res 57: Kawaguchi M, Yanokura M, Banno K et al. Analysis of a correlation between the BRAF V600E mutation and abnormal DNA mismatch repair in patients with sporadic endometrial cancer. Int. J. Oncol. 2009; 34; Kitayama J, Longacre TA, Lious S, Ma L, Arber D, Pai RK. MLH1deficient ovarian and endometrial carcinomas most often result from epigenetic silencing of MLH1 by promoter hypermethylation and do not harbor BRAF V600e mutations: implications for identifying patients with Lynch syndrome (LS). Mod. Pathol. 2011; 24; 252A. Peltomäki P, Vasen H. (2004). Mutations associated with HNPCC predispositionupdate of ICGHNPCC/Insight mutation database. Dis Markers20: Robertson KD. DNA methylation and human disease. Nature Reviews Genetics, 2005; 6, Samowitz WS, Albertsen H, Herrick J, et al. Evaluation of a large, populationbased sample supports a CpG island methylator phenotype in colon cancer. Gastroenterology. 2005;129(3): Suraweera N., Duval,A., Reperant,M., Vaury,C., Furlan,D., Leroy,K., Seruca,R., Iacopetta,B. and Hamelin,R. (2002) Evaluation of tumor microsatellite instability using five quasimonomorphic mononucleotide repeats and pentaplex PCR. Gastroenterology, 123, Varley KE, Mutch DG, Edmonston TB, Goodfellow PG and Mitra RD. Intratumor heterogeneity of MLH1 promoter methylation revealed by deep single molecule bisulfite sequencing Nucl. Acids Res. (2009)