Test Id : MTNON

When this test is ordered, slide review will always be performed at an additional charge to ensure specimen adequacy.

This assay provides a genome-wide methylation profile and MGMT promoter methylation status of primary central nervous system (CNS) tumors.

For next-generation sequencing to evaluate for microsatellite instability status, somatic mutations, and rearrangements involving targeted genes associated with CNS tumors, order NONCP / Neuro-Oncology Expanded Gene Panel with Rearrangement, Tumor.

For evaluation of copy number abnormalities and loss of heterozygosity, order CMAPT / Chromosomal Microarray, Tumor, Formalin-Fixed Paraffin-Embedded.

Multiple oncology (cancer) gene panels are available. For more information see Hematology, Oncology, and Hereditary Test Selection Guide.

Pathology report (final or preliminary), at minimum containing the following information must accompany specimen for testing to be performed:

1. Patient name

2. Block number-must be on all blocks, slides, and paperwork (can be handwritten on the paperwork)

3. Tissue collection date

4. Source of the tissue

5. Pathologic diagnosis (final or preliminary)

This assay requires at least 60% tumor nuclei.

-Preferred amount of tumor area with sufficient percent tumor nuclei: tissue 360 mm(2)

-Minimum amount of tumor area: tissue 144 mm(2)

-These amounts are cumulative over up to 20 unstained slides and must have adequate percent tumor nuclei.

-Tissue fixation: 10% neutral buffered formalin, not decalcified 

-For specimen preparation guidance, see Tissue Requirements for Solid Tumor Next-Generation Sequencing. In this document, the sizes are given as 6 mm x 6 mm x 10 slides as preferred: approximate/equivalent to 360 mm(2) and the minimum as 4 mm x 4 mm x 10 slides: approximate/equivalent to 144 mm(2).

-If ordered in conjunction with other tests, please refer to specimen requirements for the specific tests.

Preferred:

Specimen Type: Tissue block

Collection Instructions: Submit a formalin-fixed, paraffin-embedded tissue block with an acceptable amount of tumor tissue

Acceptable:

Specimen Type: Tissue slide

Slides: 1 Hematoxylin and eosin-stained and 20 unstained

Collection Instructions:

Submit the following slides:

1 Slide stained with hematoxylin and eosin

AND

20 Unstained, nonbaked slides with 5-micron thick sections of the tumor tissue

Note: The total amount of required tumor nuclei can be obtained by scraping up to 20 slides from the same block.

Additional Information: Unused unstained slides will not be returned.

• Tissue Requirements for Solid Tumor Next-Generation Sequencing

If not ordering electronically, complete, print, and send an Oncology Test Request (T729) with the specimen.

See Specimen Required

Assisting in tumor profiling by identifying methylation family and class that may support a diagnosis or help determine prognosis for patients with central nervous system tumors

This test is not intended for use in hematologic malignancies.

This test uses a methylation array to evaluate genome-wide methylation of central nervous system (CNS) tumors. Results are analyzed using the NCI/Bethesda CNS tumor classifier v2.0 developed by the National Institutes of Health, an in-house nearest-neighbors assisted unsupervised analysis (NN Method), and the MGMT promoter methylation mgmtstp27 R package.

When this test is ordered, slide review will always be performed at an additional charge to ensure specimen adequacy.

Methylation array profiling has shown clinical utility in establishing and refining the classification of central nervous system (CNS) tumors and has been incorporated as either essential or desirable diagnostic criteria for over 50% of tumor types included in the 2021 5th World Health Organization (WHO) classification of CNS tumors. Two specific tumor types, diffuse glioneuronal tumor with oligodendroglioma-like features and nuclear clusters and high-grade astrocytoma with piloid features, are definitively diagnosable only with the use of methylation profiling. The c-IMPACT-NOW working group (involved in planning of future WHO classifications) recently codified the utility of DNA methylation profiling as recommended for diagnostic practice, particularly for difficult-to-diagnose CNS tumors. MGMT promoter methylation status is also obtained using this test and is a well-established prognostic biomarker and a strong predictor of alkylating chemotherapy response for patients with glioblastoma, IDH-wildtype. MGMT promoter methylation testing using methylation array has been clinically validated in retrospective and prospective studies.

An interpretive report will be provided.

The interpretation of molecular biomarker analysis includes an overview of the results and the associated diagnostic implications.

This assay has three reportable results:

1) Tumor classification at methylation family and methylation class levels generated by the NCI/Bethesda Classifier v.2.0, reported as shown in Table 1

2) Nearest neighbor Method (NN Method) class compared to the NCI/Bethesda Classifier v.2.0 methylation class results, reported as shown in Table 1

3) MGMT promoter methylation status, reported as methylated (score > or =0.358) or unmethylated (score <0.358)

Table 1. Classifier and Nearest Neighbor (NN) Method Interpretation

(a) NN Method Class result is identical to Classifier Class

(b) NN Method Class result is different from Classifier Class

(c) NN Method Class result was not available as a consensus class among at least three of the five closest reference dataset samples (“neighbors”) was not reached

*For Classifier Class NO MATCH, there is no reportable Classifier Class to be compared with NN Method Class

Abnormal methylation patterns sufficiently distinct from the reference dataset patterns can impact the ability of the classifier to provide high confidence results.

Discordant methylation profiling results may be obtained using different classifiers and different classifier versions.

The nearest-neighbors assisted unsupervised analysis (NN) method is complementary to the classifier and the results are not to be used as a stand-alone method.

Discordant MGMT methylation results may occur due to different testing methodologies and/or evaluation of different CpG sites.

Performance Characteristics

Validation studies demonstrated overall concordance of results between samples tested in-house and National Institutes of Health-tested samples was 88.7% (55/62 samples), with 91.9% (57/62) Family/Class classifier concordance, 98.4% (61/62) nearest-neighbors assisted unsupervised analysis concordance, and 98.4% (61/62) MGMT promoter methylation concordance.

Inter- and Intra-run reproducibility replicates were 100% concordant at limit of detection for all reportable results.

To ensure accuracy, this test will be performed on cases estimated by a pathologist to have at least 60% tumor cells and requires a minimum of 75ng of DNA.

1. WHO Classification of Tumours Editorial Board. Central nervous system tumours. 5th ed. World Health Organization; 2021. WHO Classification of Tumours. Vol 6

2. Aldape, K, Capper D, von Deimling A et al. cIMPACT-NOW update 9: Recommendations on utilization of genome-wide DNA methylation profiling for central nervous system tumor diagnostics. Neurooncol Adv. 2025:7(1): vdae228

3. Bady P, Delorenzi M, Hegi ME. Sensitivity analysis of the MGMT-STP27 model and impact of genetic and epigenetic context to predict the MGMT methylation status in gliomas and other tumors. J Mol Diagn. 2016;18(3):350-361

4. Bady P, Sciuscio D, Diserens AC, et al. MGMT methylation analysis of glioblastoma on the Infinium methylation BeadChip identifies two distinct CpG regions associated with gene silencing and outcome, yielding a prediction model for comparisons across datasets, tumor grades, and CIMP-status. Acta Neuropathol. 2012;124(4):547-560

5. Capper D, Jones DTW, Sill M, et al. DNA methylation-based classification of central nervous system tumours. Nature. 2018;555(7697):469-474

6. Capper D, Stichel D, Sahm F, et al. Practical implementation of DNA methylation and copy-number-based CNS tumor diagnostics: the Heidelberg experience. Acta Neuropathologica 2018; 136(2):181-210

7. Furtado L, Ikemura K, Benkli C, et al. General applicability of existing CAP accreditation requirements to clinical implementation of machine learning-based methods in molecular oncology testing. Arch Pathol Lab Med. 2025;149(4):319–327

8. Galbraith K, Vasudevaraja V, Serrano J, et al. Clinical utility of whole-genome DNA methylation profiling as a primary molecular diagnostic assay for central nervous system tumors-A prospective study and guidelines for clinical testing. Neurooncol Adv 2023;5(1):vdad076. (In eng). doi:10.1093/noajnl/vdad076

9. Kling T., Wenger A., Beck S., Caren H. Validation of the MethylationEPIC BeadChip for fresh-frozen and formalin-fixed paraffin embedded tumours. Clinical Epigenetics 2017;9:33

10. Wu Z, Abdullaev Z, Pratt D, et al. Impact of the methylation classifier and ancillary methods on CNS tumor diagnostics. Neuro Oncol. 2022;24(4):571-581. doi:10.1093/neuonc/noab227

This test uses the Illumina Infinium MethylationEPIC v2.0 kit to detect cytosine methylation at 935,000 CpG loci based on highly multiplexed genotyping of bisulfite-converted gDNA (Package insert: Infinium MethylationEPIC v2.0 BeadChip, Illumina Inc; 2022).

Sample data are analyzed using an in-house developed quality control module, the proprietary National Institutes of Health-developed NCI/Bethesda CNS (central nervous system) tumor classifier v2.0 and an in-house developed nearest-neighbors assisted unsupervised analysis (NN method).(Wu Z, Abdullaev Z, Pratt D, et al. Impact of the methylation classifier and ancillary methods on CNS tumor diagnostics. Neuro Oncol. 2022;249[4]:571-581. doi:10.1093/neuonc/noab227]) The NCI/Bethesda classifier generates tumor classification at family and class levels. It also provides a Uniform Manifold Approximation and Projection (UMAP) plot to visualize how the tested sample clusters relative to the reference dataset. Since UMAP is inherently subjective, we implemented the NN method to identify the five closest reference samples ("neighbors") to the test sample, enabling objective estimation of the test sample methylation class cluster. The NN method is complementary to the classifier and interpreted in the context of classifier results.

The MGMT promoter methylation status is predicted using the mgmtstp27 R package.(Bady P, Sciuscio D, Diserens AC, et al. MGMT methylation analysis of glioblastoma on the Infinium methylation BeadChip identifies two distinct CpG regions associated with gene silencing and outcome, yielding a prediction model for comparisons across datasets, tumor grades, and CIMP-status. Acta Neuropathol. 2012;124[4]:547-560. doi:10.1007/s00401-012-1016-2)

A pathology review and macro dissection to enrich for tumor cells is performed prior to slide scraping.

Varies

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This test was developed and its performance characteristics determined by Mayo Clinic in a manner consistent with CLIA requirements. It has not been cleared or approved by the US Food and Drug Administration.

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