Specimen requirements

Formalin Fixed Paraffin Embedded (FFPE) Tissue:

A minimum of 10 unstained slides of ~5um thickness paraffin embedded tissue;
1 H&E stained slide with the tumour area of interest clearly marked by a histopathologist - avoid/exclude any normal tissue, mucin or necrotic material from marked tumour areas;
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.
Minimum tumour load required in the marked area is 30%. Specimens with tumour loads below this limit prevent accurate assessment of methylation levels and will not be accepted for analysis;
The request form must have two forms of identification matching the slides received. The entire block number must be included on the request form. Failure to meet there requirements will delay results;
A copy of the complete pathology report on the sample block received is required, including tumour origin/diagnosis (colorectal or endometrial carcinomas), and MMR status (IHC) showing loss of MLH1 and PMS2 protein expression;
For normal tissue testing: please send 1 H&E stained slide plus 6 unstained sides with normal tissue 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.
Slides are to be kept at room temperature at all times.

Turnaround Time: 3 weeks
Assay Method
Agena EpiTYPER MassARRAY

The methylation status of the promoter region of the ML H1 gene is established by means of Agena Bioscience's EpiTYPER MassARRAY System, using base-specific cleavage and MALDI-TOF Mass Spectrometry. This method is performed following DNA extraction from paraffin embedded tissue and treatment with bisulfite. The following thresholds are used: unmethylated (0-10%); equivocal (11-20%); methylated (>20%). Locus Reference Genomic: LRG_216t1.

Diagnostic Use and Interpretation
MLH1 promoter methylation analysis is useful to distinguish sporadic from inherited colorectal (CRC) and endometrial cancers in tumours that are MLH1-deficient by IHC staining and/or have high levels of microsatellite instability (MSI-H). The majority of MSI in sporadic cases of CRC/endometrial cancers is caused by loss of expression of MLH1 by promoter hypermethylation, whilst hypermethylation is rare in inherited cases. The absence of MLH1 promoter hypermethylation is associated with an increased likelihood of a germline mutation in a DNA mismatch repair gene. Normal tissue testing can also be performed in order to exclude the possibility of constitutional epivariants of the MLH1 gene. MLH1 promoter hypermethylation analysis results should be considered with other clinical risk factors when determining Lynch Syndrome likelihood.

Please note: this Lynch syndrome testing pathway has only been stablished for colorectal and endometrial cancers at this time. Currently, the clinical utility of the MLH1 methylation testing on alternative tumour types is unknown, therefore results from non-CRC/endometrial tumours will not be interpreted.

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.

References
Bock et al 2016 Nature Biotechnology 34, 726-737
Bruegl, Et al 2014 Curr Pharm Des.; 20(11): 1655-1663.
Claus R1 et al 2012 Epigenetics.(7):772-80.

Contact Information
Diagnostic Genetics: 307 4949 (extension 22008)

Aline Marubayashi: alinem@adhb.govt.nz

Chloe Pearson: cpearson@adhb.govt.nz

Specimen Transport Instructions for Referring Laboratories
Please send all requests to the histology laboratory holding the FFPET block (for slide preparation & histopathologist assessment):

1. For FFPET samples NOT held at LabPLUS: Please send the request form and instructions to the histology laboratory holding the block.

2. For FFPET samples held at LabPLUS: Please send the request form and instructions to LabPLUS Histology Department :

Contacts: Tania Smith ( tanias@adhb.govt.nz ); Tanya Fulton ( tanyaf@adhb.govt.nz ); Debbie O'Regan ( debbieo@adhb.govt.nz )

LabPLUS address:

Specimen Reception, LabPLUS

Building 3, Auckland City Hospital

Gate 4 off Grafton Rd

Grafton

Auckland 1148

New Zealand

Last updated at 11:15:49 08/04/2024