Molecular Pathology

These tests either aid the histological diagnosis of brain tumours or are essential for planning enrolment into clinical trials or to decide for the optimum therapy. We offer the tests to all NHS (Provider-to-provoider NHS price see below) and private hospitals . Price list (NHS) and typical turnaround times are at the end of this website.

Important information regarding enquiries: Please avoid contacting the molecular pathology team or our office to enquire about individual results before the expected turnaround times.  Please first contact your local Pathology team to enquire when the sample was referred out, and when it was received by us (your pathology team will have received a receipt). Refer to the turnaround times below (see list at the end of this website).

Important: This request form is updated regularly. It is valid only on the day of download. Please regularly download new copies of this form

basic guidelines for the combination of biomarkers in CNS gliomas

wallchart: molecular diagnostic algorithm of adult gliomas and ependymal tumours

Further inquiries about our services and referrals:
Prof Sebastian Brandner
sebastian.brandner@nhs.net

Dr Zane Jaunmuktane
zane.jaunmuktane@nhs.net 

Dr Ashirwad Merve
amerve@nhs.net

Technical inquiries, specimen submission: 
UCLH.molecular.neuropathology@nhs.net

Monika Dutt monika.dutt@nhs.net 
Mojda Mushfiq mojda.mushfiq@nhs.net 
Jack Grant, jack.grant5@nhs.net 
Viassa Rampersad, v.rampersad1@nhs.net
UCLH.molecular.neuropathology@nhs.net

Clinical bioinformatics
Rossella Rispoli r.rispoli@ucl.ac.uk 
 

Molecular test portfolio for gliomas

The main differential diagnosis of gliomas is to distinguish astrocytomas, oligodendrogliomas and IDH wild-type glioblastoma. These require a set of markers which we offer as "package". This allows us to return a precise integrated diagnosis which combines histological assessment, immunohistochemical testing with established biomarkers, supplemented with molecular tests which assess point mutations and chromosomal losses and gains.

Methylome analysis for refined diagnosis of adult brain tumours 

Characteristic patterns of DNA methylation of CpG islands in the genome are key features in various tumours. The Illumina 850k EPIC array assesses the DNA methylation status of a small proportion of the 850,000 individual CpG sites on the array. An algorithm, developed at the DKFZ in Heidelberg, Germany, is able to distinguish brain tumour entities based on their methylation profile. It is currently based on over 10,000 reference brain tumour cases and is periodically updated.

Expected turnaround time: between 10 and 20 days from arrival of referral in our department

Guidance when to use the classifier: in adult brain tumours, we recommend using the classifier for all histologically unclear CNS neoplasms. In particular, when a basic molecular pathology workup, for example using the above markers, does not yield diagnostically relevant results. We also recommend using the classifier for comprehensive analysis of all astrocytomas and oligodendrogliomas, and for risk stratification of meningiomas, in particular for atypical, unusual and malignant meningiomas.  

As an informal (i.e. not evidence-based) practical guidance, we consider using the Illumina array when a CNS neoplasm cannot be diagnosed with approximately 8-10 immunostains, and/or with any of the above tests. 

With small biopsies, we recommend considering the Illumina array well before the tissue is exhausted.

The following table lists typical examples of possible combinations of tests and their minimum cost. All prices are "Provider-to-Provider" between NHS Trusts. The prices include interpretation and evaluation of the neoplasms in the context of the referred diagnosis, our own opinion based on H&E and the molecular test result. For a formal opinion on the histological diagnosis, we recommend referral with all IHC Slides and a specific request in the referral letter, but please also use our referral form. 
The prices are for guidance only and can vary depending on the number of blocks referred, extent of consultation and repeat tests due to reduced sample quality. Referral of multiple blocks can increase the cost of our tests.

Molecular analysis of LOH1p/19q in astrocytomas and oligodendrogliomas

Relevance: Loss of 1p/19q is a feature of Oligodendrogliomas. 1p/19q loss is an early event in oligodendroglioma and is retained during tumour progression.
The combined loss of 1p/19q is related to survival. This improved prognosis is present whether observed at initial diagnosis or at recurrence. 1p/19q loss oligodendrogliomas also progress more slowly at recurrence.
CDKN2A/B homozygous deletion is a prognostic marker for IDH-mutant astrocytomas. A deletion indicates more rapid progression and recurrence.
This test is done in conjunction with copy number assays for 7p (EGFR locus), and sequencing of the following genes: IDH1/IDH2, Histone H3.3 (G34 and K27), BRAF and TERT promoter. 

Test Methods:

We recommend methylation array with concomitant copy number assay.

Expected turnaround time: approximately 10-15 days from arrival of referral in our department

Molecular analysis of EGFR amplification and EGFRvIII mutations 

Test Method:
EGFR: Copy Number Assay with Real-Time PCR. If EGFR is not amplified, EGFRvIII will not be tested 

Expected turnaround time: approximately 20 days from arrival of referral in our department

Relevance:

• EGFR amplification: Approximately 50% of GBMs are characterised by overexpression of EGFR and EGFR gene amplification.
• EGFRvIII enhances the tumourigenic potential of GBM by activating and sustaining mitogenic, anti-apoptotic and pro-invasive signalling pathways. EGFRvIII is a tumour-specific mutation that is widely expressed in GBM and other neoplasms, resulting from an in-frame deletion of 801 bp spanning exons 2 to 7 of the coding region of EGFR. This in-frame deletion mutation splits a codon, resulting in a novel glycine at the fusion junction producing a tumour-specific epitope target for cellular or humoral immunotherapy. 

Method: 

EGFR: Copy Number Assay with Real-Time PCR.
EGFRvIII mutations: RNA isolation from Paraffin sections, Reverse transcription and detection of the fusion gene by Sequencing.  

BRAF fusion gene and V600E point mutations

Relevance: The identification of the BRAF a fusion gene (A tandem duplication occurring at 7q34 leading to a fusion between KIAA1549 and BRAF genes) in more than 60% of Pilocytic astrocytomas, but not in morphologically similar neoplasms or in reaction lesions is used for molecular diagnostics for PA. The BRAF gene resides on chromosome 7q, near the HIPK2 locus and has been linked to numerous other cancers. 

The V600E mutation is located in exon 15 of the BRAF gene. 66% WHO grade II pleomorphic xanthoastrocytomas and WHO grade III PXA with anaplasia, 18% of WHO grade I gangliogliomas and in 9% pilocytic astrocytomas (strongly associated with extra-cerebellar location). This makes the V600E test a useful diagnostic marker for these. 

Method:
BRAF Fusion: RNA -based Next generation sequencing (part of a panel). Test performed at the Royal Marsden Hospital, North Thames Genome Laboratory Hub. 

Expected turnaround time: approximately 21 days from arrival of referral in our department

Methylation of the MGMT promoter in gliomas

Relevance: MGMT (O6-methylguanine DNA methyltransferase) is a key enzyme in the DNA repair network. MGMT removes the mutagenic and cytotoxic adducts from O6 guanine in DNA, the preferred point of attack of alkylating chemotherapeutic agents (i.e. BCNU, temozolomide, etc). Hypermethylation of the CpG island located in the promoter region of MGMT is primarily responsible for the loss of MGMT function in many tumour types. MGMT hypermethylation alone, without treatment with an alkylating agent, is a negative prognostic marker. MGMT hypermethylated tumours are more sensitive to the killing effects of alkylating drugs used in chemotherapy.

Test Method: Methylation-sensitive High-Resolution Melting (MS-HRM) analysis.

Expected turnaround time: approximately 15 days from arrival of referral in our department, if ordered as an individual test