MLH1 Hypermethylation Testing (FFPE Tissue)

Testing Laboratory

Auckland Te Toka Tumai | Auckland Hospital

Department

Diagnostic Genetics

Delphic Registration Code (Alpha Code)

DGEN

Test Code (Numeric Code)

4218

Laboratory Handling

Transport

Slides must be securely packaged to prevent breakage or loss during transport. Specimens can be transported at room temperature in the range of 8 Degrees Celsius to 24 Degrees Celsius.

Registration

Please store samples at room temperature. Only place barcode labels on slide boxes; do not add barcode labels to individual slides. Please scan all referral paperwork and the send original referral with the sample.

Laboratory

Diagnostic Genetics - Molecular Oncology

Sample Stability

Keep samples at room temperature.

Synonyms

MLH1 Methylation Testing, MLH1 gene promoter methylation testing, MLH1, Lynch syndrome

Turnaround Time

3 weeks

Specimen Type

• Tissue

Specimen Requirements

Tube/Container Type:

Preferred

• Please refer to Additional Information below
• Slide(s)

Paediatric Specimen Requirements

Tube/Container Type:

Preferred

• Please refer to Additional Information below
• Slide(s)

Pre-testing Requirements

If you are a requesting clinician (e.g. oncologist), please contact the histology laboratory holding the FFPE block to arrange processing and submission of the specimen.

Additional Specimen Information

1. Unstained slides
Fine-needle aspirates, core biopsies and small/low-cellularity samples: a minimum of 10 unstained FFPE slides (5 micron sections)
Larger resections: a minimum of 6 unstained FFPE slides (5 micron sections)

2. An H&E slide clearly targeted by a histopathologist
Please include the tumour content assessed as this is an essential requirement for testing. Samples without a clearly marked target AND tumour % will not be processed.
The target for macrodissection should contain at least 30% of viable tumour cells in a background of no more than 70% normal tissue content, including inflammatory cells - avoid including necrotic material in the targeted area. Slides are to be kept at room temperature at all times.
For tumours with heterogeneous/subclonal IHC MLH1/PMS2 loss, please target regions showing loss only (avoid proficient areas).

3. Each slide must be clearly labelled with at least two patient identifiers (e.g., name and block number, or NHI and block number).

4. Request forms must include the sample block number, any relevant clinical details and tumour diagnosis. Additionally, the request form must include the IHC MMR status, showing loss of MLH1 and PMS2.

5. If normal tissue testing is also required, please follow the sample slide requirements above. Note: preferably the normal tissue should be collected from an area far from tumour, as methylation levels are commonly altered on normal cells surrounding the tumour area and may not be representative of normal tissue.

Specimens arriving with incomplete labelling/documentation or incorrect sample requirements will be rejected.

Aliquot Instructions

Transport to Lab

Ambient (8 - 24 degrees Celsius)

Test Method

The methylation status of the MLH1 promoter region is assessed by Bio-Rad droplet digital PCR (ddPCR) following bisulfite conversion of tumour-derived DNA. This method provides a quantitative assessment of methylation levels, which are then interpreted and reported qualitatively.

The workflow utilizes two separate duplex ddPCR probe assays, each incorporating specific probes for both methylated and unmethylated MLH1 promoter sequences. Together, these assays target a total of 3 CpG* units correlated to gene expression, allowing for the precise calculation of a fractional abundance (methylation fraction) for each sample.

*GRCh37 coordinates (NM_000249.4): 3:37034814, 3:37034825 and 3:37034840 (-204, -193 and -178 relative to the transcription start site) [1-2]

Limitations / Interference

Test results can be affected by factors including: intra-tumoral methylation heterogeneity, the presence of normal tissue contamination of the tumour sample (or tumour contamination on a normal tissue sample), PCR reaction inhibitors (mucin, necrotic material), or quality of the specimen. Therefore, the clinical diagnosis or therapy should not be solely based on this assay and the results should be considered in conjunction with clinical information and additional diagnostic tests.

Uncertainty of Measurement

Reflecting a 95% confidence interval (k=2), the analytical uncertainty figures for this assay are defined by interpretive range: • Methylated Range (≥10% fractional abundance): UOM is <10% (calculated as 2 x %CV). Note: expressed in relative units. • Unmethylated & Equivocal Ranges (<10% fractional abundance): UOM is <2% absolute fractional abundance (calculated as 2 x SD). Note: expressed in absolute units rather than %CV due to stochastic variation inherent to low target concentrations.

1. Hampel H et al. Assessment of tumor sequencing as a replacement for Lynch syndrome screening and current molecular tests for patients with colorectal cancer. JAMA oncology. 2018 Jun;4(6):806-13. (PMID: 29596542)
2. Crosbie EJ, Kitson SJ, McAlpine JN, Mukhopadhyay A, Powell ME, Singh N. Endometrial cancer. The Lancet. 2022 Apr 9;399(10333):1412-28. (PMID: 35397864)
3. Helderman NC et al. MLH1 promotor hypermethylation in colorectal and endometrial carcinomas from patients with Lynch syndrome. The Journal of Molecular Diagnostics. 2024 Feb 1;26(2):106-14. (PMID: 38061582)
4. National Comprehensive Cancer Network (2025). NCCN Clinical Practice Guidelines in Oncology: Colorectal/Familial High-Risk Assessment: Colorectal, Endometrial, and Gastric (Version 1.2025, cited 2026 Jun 03). Available from: https://www.nccn.org/professionals/physician_gls/pdf/genetics_ceg.pdf?utm_source=openevidence#page=35
5. Deng G, Chen A, Hong J, Chae HS, Kim YS. Methylation of CpG in a small region of the hMLH1 promoter invariably correlates with the absence of gene expression. Cancer Res. 1999;59(9):2029–2033. (PMID: 10232580)
6. Benhamida JK, Hechtman JF, Nafa K, et al. Reliable clinical MLH1 promoter hypermethylation assessment using a high-throughput genome-wide methylation array platform. J Mol Diagn. 2020;22(3):368–375. (PMID: 31881335)

Contact Information