CNS Tumour Molecular Testing

Summary

Following neuropathology assessment, glioma samples from patients of all ages can be stratified for molecular testing depending on tumour grade.

Mutation testing by next generation sequencing can be undertaken for any combination of IDH1, IDH2, BRAF, histones H3F3A and HIST1H3B, EGFR, and TERT promoter.

1p/19q and EGFR amplification status is tested by FISH.

MGMT methylation status is assessed by methylation specific pyrosequencing.

KIAA1549-BRAF fusion is assessed by RT-PCR.

The data below summarises the testing strategy:

FFPE sample requirements

Fresh tissue for cell culture is no longer necessary for molecular testing. All molecular investigations are performed on formalin fixed paraffin wax embedded tissue (FFPE).

For all samples, please send either a paraffin block which we will cut and mark for testing, or send two H&E stained slides (labelled front and back) with tumour material marked, along with additional slides from a region of the tumour that corresponds with the tissue between the H&E labelled slides. We will be guided by this to determine which tissue area to analyse.

If extracted DNA is to be sent for testing, ~15ul of 10ng/ul DNA will usually be sufficient for DNA based testing. If choosing to use this option, please liaise directly with the genetics laboratory beforehand to establish requirements.

• Gliomas for 1p19q assessment – Two labelled H&E stained slides are required as above, plus 4 sections of 4µm thickness for 1p/19q FISH studies. Slides for FISH studies should be coated and positively charged.
• Gliomas for mutation studies and/or MGMT methylation studies – Two labelled H&E stained slides are required as above, plus 10 sections (these don’t need to be coated or positively charged) of 4µm thickness for IDH/BRAF/TERT/H3F3A/HIST1H3B mutation studies and MGMT testing.
• For selected low grade tumours – Two labelled H&E stained slides are required as above, plus 20 sections of 4µm thickness (not coated or positively charged) for BRAF-KIAA1549 fusion testing by RNA analysis. Special precautions should be in place to reduce the risk of contamination of RNases. Gloves should worn at all times when handling FFPE blocks/sections for RNA work.

Please use clear patient identifiers on the sample containers, slides and referral form.

All patient samples must be labelled with at least three patient identifiers.

Please use clear patient identifiers on the sample containers, slides and referral form.

All patient samples must be labelled with at least two patient identifiers.

1p/19q testing

• 1p/19q testing is performed by FISH studies using 1p36/1q25 and 19p13/19q13 dual colour probe sets.
• 100 cells are analysed for evidence of 1p/19q co-deletion.
• Deletions are reported when seen in at least 25% of the cells analysed.
• 1p/19q testing is now required for the WHO 2016 CNS tumour classification.

References:

1. C Horbinski et al. Brain Pathology (21):57-73, 2011
2. Louis, D. N., 2016. The 2016 World Health Organization Classification of Tumours of the Central Nervous System: a summary. Acta Neuropathol, 131:803-820

IDH mutation testing

• Targeted IDH1 and IDH2 mutation analysis is performed by next generation sequencing using the Illumina MiSeq platform.
• The assay will only detect mutations affecting codon 132 of the IDH1 gene and codon 172 of the IDH2 gene.
• Over 95% of mutations detected are in IDH1 and 2-5 % in IDH2.
• Of those mutations reported in IDH1 approximately 90% occur as a result of a G to A transition at cDNA position 395, resulting in the substitution of Arginine with Histidine.
• The test will detect all known single base pair mutations within codon 132 of the IDH1 gene and codon 172 of the IDH2 gene.
• The sensitivity of the test is ~5% (ie mutations present in less than 5% of the material tested may be missed).
• IDH mutation testing is now required as part of diagnostic testing in accordance with the WHO 2016 CNS tumour classification.

References:

1. Ichimura, K. Brain Tumour Pathology 29(3):131-139, 2012
2. Louis, D. N. Acta Neuropathol, 131:803-820, 2016

BRAF mutation testing

• Targeted BRAF mutation analysis is performed by next generation sequencing using the Illumina MiSeq platform.
• The assay will only detect BRAF codon 600 cDNA positions c.1798, c.1799 and c.1800 for mutations and BRAF codons 597, 598, 599 for insertions.
• BRAF mutations are seen in a variety of gliomas, including pilocytic astrocytoma, pilomyxoid astrocytomas, pleomorphic xanthoastrocytoma, ganglioglioma, diffuse astrocytomas and occasional high grade gliomas (most usually epitheloid glioblastomas) (Refs: Horbinski et al 2013; Brandner and Deimling 2015; Brander and Jaunmuktane 2017).
• The sensitivity of the test is ~5% (ie mutations present in less than 5% of the material tested may be missed).

References:

1. Brandner and Deimling. Neuropath and Applied Neurobiol 41, 694-720, 2015
2. Horbinski et al J. Neuropathol Exp Neurol 72(1):2-7, 2013
3. Brandner and  Jaunmuktane J Neurol Nov 2, 2017

TERT promoter mutation testing

• Targeted TERT promoter mutation analysis is performed by next generation sequencing using the Illumina MiSeq platform.
• The assay will only detect TERT promoter mutations at positions c.-124 and c.-146.
• In grade II and III gliomas a TERT promoter mutation when accompanied by an IDH mutation is associated with a better outcome than wildtype tumours (Yang, P et al and Eckel-Passow J et al).
• The presence of a TERT promoter mutation in IDH wild type glioma is associated with a poorer outcome than TERT promoter and IDH wildtype tumours (Killela et al 2014; Pekmezci et al 2017).
• The sensitivity of the test is ~5% (ie mutations present in less than 5% of the material tested may be missed).

References

1. Yang, P et al. Neuro-Oncology 18(8):1099-1108, 2016
2. Eckel-Passow J et al. N Engl J Med 372;26:2499-2508, 2016
3. Killela et al. Onco target 5:6:1515-1525, 2014
4. Pekmezci et al Acta Neuropathol 133:1001-1016, 2017

Histone mutation testing

• Targeted mutation analysis of histones H3F3A and HIST1H3B is performed by next generation sequencing using the Illumina MiSeq platform.
• The assay will only detect histone H3F3A codon 28 cDNA position c.83 and  histone HIST1H3B codon 28 cDNA position c.83 for mutations.
• Mutations are described as K27M, G35R and G35V.
• Histone mutations predominantly occur in children in midline gliomas although they are also seen in other high grade gliomas.
• Histone codon 28 mutations are most commonly seen in midline gliomas and the WHO 2016 classification of a diffuse midline glioma, H3 K27M mutant.
• The histone H3F3A codon 35 variant (G35R) has been reported in a molecularly distinct  group of high grade gliomas (located outside the midline), including  histologically characterised GBM-like tumours preferentially occurring in adolescents and young adults (Sturm et al, 2012; Korshunov et al, 2016).
• The sensitivity of the test is ~5% (ie mutations present in less than 5% of the material tested may be missed).

References:

1. Kallappagoudar S et al. Chromosoma 124:177–189, 2015
2. Sturm et al, Cancer Cell: 425-437, 2012
3. Korshunov et al, Acta Neuropathol: 137-146, 201 

Other mutation testing

• Requests for the lab to develop testing for mutations in other genes will be considered in future development planning.

MGMT methylation testing

• MGMT methylation testing is performed by pyrosequencing on grades II-IV  gliomas.
• This assay detects methylation at the 8 CpG islands within c.15-58 of the MGMT promoter. Reference sequence NM_002412.3.
• A normal result does not guarantee MGMT methylation is absent. It is assumed that the material tested is of tumour origin and relies on the presence of sufficient tumour cells, however contamination from normal tissue cannot be excluded.
• An average methylation across all 8 CpGs tested of >15% is reported as methylated, between 10 and 15% is reported as equivocal and <10% as unmethylated.
• These cut-offs are currently being reviewed in the light of local data and are likely to change in the near future.
• MGMT methylation in glioblastoma is associated with improved prognosis and an improved response to alkylating chemotherapy (Ref: Stupp R et al). Recent studies have shown that in IDH wild type patients, this survival advantage is greater in those that also have a TERT promoter mutation (Ref Nguyen et al 2017).
• MGMT methylation in glioblastoma in elderly patients is associated with improved overall and progression free survival (Refs: Gallego Perez-Larraya et al and Wick et al).
• Patients with MGMT unmethylated have shown a poorer survival than those with MGMT methylation (Ref Nguyen et al 2017). This study has also shown that the poorest survival is in those patients with IDH wild type and with a TERT promoter mutation.
• MGMT methylation in anaplastic oligodendroglioma/astrocytoma is associated with a longer progression free survival (Refs Wick et al and van den Bent et al).

References:

1. Quillien et al. Cancer 4201-4211, 2015
2. Quillien et al. J Neuroncol 116:487-496, 2014
3. Stupp R et al. Lancet Oncol 10:459-66, 2009
4. Christians et al. MPLoS ONE7(3): e33449, 2012
5. Gallego Perez-Larraya et al. Lancet Oncol 13:707-715, 2012
6. Van den Bent et al. J Clin Oncol 27:5881-5886, 2009
7. Nguyen et al. Neuro-Oncology 19(3), 394–404, 2017

KIAA1549-BRAF fusion testing

KIAA1549-BRAF fusion testing is performed for selected low grade gliomas using RT-PCR, in conjunction with a next generation sequencing based approach to BRAF mutation detection in order to provide a full BRAF service.

 A two-tiered approach is undertaken:

 1. BRAF fusion testing is performed by RT-PCR using the RotorGene-Q.

• This assay will only detect the three most common KIAA1549-BRAF fusions (>95%) between exons 16-9, 15-9 and 16-11 and is based on Tian et al. 2011. There are a number of rarer BRAF fusions that this assay does not test.
• This assay relies on the presence of sufficient tumour cells within the sample tested. A minimum sensitivity of 3.1% mutant RNA sequence against a wild-type background has been estimated from in house calculations.
• The KIAA1549-BRAF fusion gene has been detected in ~ 70% of cases pilocytic astrocytoma. Detection of the KIAA1549-BRAF fusion therefore supports a diagnosis of pilocytic astrocytoma but absence of this gene fusion does not exclude diagnosis of this tumour type (Horbinski, C. 2013). It is also seen in pilomyxoid astrocytoma.

 2. BRAF Mutation testing is performed at the same time as BRAF fusion testing, by Next Generation Sequencing, using the Illumina MiSeq platform

• Samples are either tested just for BRAF mutations, or can also be tested for mutations in IDH1, IDH2, TERT promoter, and histones using the lab’s glioma panel.
• The test will detect single base pair mutations within BRAF codon 600 cDNA positions c.1798, c.1799 and c.1800 and BRAF codons 597, 598, 599 for insertions, and mutations affecting codon 132 of the IDH1 gene and codon 172 of the IDH2 gene.
• This assay will not detect the presence of other mutations
• The sensitivity of the test is ~5% (ie mutations present in less than 5% of the material tested will be missed)

References:

1. Tian et al. J Mol Diagn, 13, 669-77, 2011
2. Horbinski, C. J Neuropathol Exp Neurol, 72(1), 2-7, 2013

Other FISH testing

Additional FISH tests can be undertaken following specific requests to assist with diagnosis and prognosis, and/or tumour grading. Requirements for FISH studies are the same as those described above for 1p19q testing. The majority of FISH probes used are obtained commercially. In cases where no commercial probes are available, it may be possible for the department to produce its own “home-grown” FISH probes.

A range of different FISH tests can be performed:

EGFR FISH testing

FISH testing for EGFR amplification can be undertaken using a single copy EGFR probe with a 7 centromere control. Histologic grade II and III IDH-wildtype diffuse astrocytic gliomas which contain high-level EGFR amplification have recently been shown to correspond to WHO grade IV tumours (Ref Brat et al, 2018). This study suggest that such tumours should be referred to as diffuse astrocytic glioma, IDH-wildtype, with molecular features of glioblastoma, WHO grade IV

 References:

1. Brat et al, Acta Neuropathol. Nov;136(5):805-810, 2018

YAP1 and RELA FISH testing

FISH testing of selected ependymomas can be arranged to detect rearrangements of the YAP1 or RELA genes. Break-apart FISH probes have been developed for this purpose

C19MC testing

FISH testing can be undertaken to assess the presence of C19MC amplification in embryonal tumours with multilayered rosettes

Other CNS tumours

Other than gliomas, a limited molecular testing service is available for other tumour types. This is mainly FISH testing:

Reporting of results

• Results are reported electronically to the referring clinician. This is either via the local LIMS or as a PDF by email. Where directly relevant, abnormal results may be telephoned prior to the electronic report being available, and the interpretation and implication discussed
• In response to telephone enquiries, only normal results or those that confirm a previous finding are given to a clinician’s secretary or the clinic sister. All other results are only given to clinicians
• Reports can be emailed if you supply the genetics laboratory with a secure NHS.net address. If no NHS net account is available, a secure route for emailing results can be set up. A paper copy will generally not be posted.

Turnaround times (working days)

• FISH: 2-5 days
• IDH/BRAF/TERT/Histones: 5-10 days
• MGMT: 5-10 days
• BRAF fusion: 5 working days
• Priority can be adjusted in line with clinical urgency and the need to present at MDT meetings.
• Most urgent FISH results can be reported within 2-3 days of sample receipt.

In development

We are currently looking at expanding the range of genes tested by NGS to ensure the lab can offer all the tests described in the NHSE national cancer test directory published in 2018 – https://www.england.nhs.uk/publication/national-genomic-test-directories/.

Please contact the laboratory for any further information or to request a test not listed.

Sample Transport

All requests for molecular testing (including IHC, FISH, RT-PCR, and sequencing) should be sent to the laboratory. For the laboratory address and contact details for sending samples and enquiries please see the laboratory contacts page.