Royal Prince Alfred Hospital Royal Prince Alfred Hospital
Department of Neuropathology

Molecular Neuropathology

Ò NEUROPATHOLOGY – Molecular Request Form (Version 4.1)  (updated 15/05/2020)


The Molecular Neuropathology Laboratory offers comprehensive NATA-accredited molecular testing for brain tumour specimens. We provide reliable, timely and appropriate services to patients.

Effective from 1 May 2020, some of the molecular diagnostic tests offered are included in the Medicare Benefit Scheme (MBS) for eligible patients.

Please refer to our request form or contact us for further details.

 

 

Next Generation Sequencing (NGS) Glioma Panel

The Next Generation Sequencing (NGS) glioma panel provides NATA accredited service for the assessment of relevant glioma-associated molecular alterations.

NATA accredited panel targets

Single Nucleotide Variation (SNV) for

Copy Number Variation (CNV) for

IDH1 (codon 132)
IDH2 (codon 172)
TERT promoter (C228T, C250T)
H3F3A (codons 27,34)
BRAF (codon 600)

1p
19q
EGFR

Detection of IDH1/2 pathological variant status (MBS item 73372) and chromosome 1p19q co-deletion (MBS item 73371) by NGS will be partially covered under MBS for eligible patients.

Additional genes are in the process of validation for NATA accreditation. Once accredited they will be added to our NATA accredited panel targets. At present, the results of the unaccredited component can only be released on request as “research use only” results, and cannot be used for clinical decision making.

Research panel targets (red-coloured genes for SNV & CNV)

ATRX, CDKN2A, CDKN2B, CDKN2C, CIC, EGFR, FUBP1, HIST1H3B, HIST1H3C, KIAA1549, MYC, MYCN, NF1, NF2, NRAS, PDGFRA, PIK3CA, PIK3R1, PTEN, RB1, TP53, chr7 gain, chr10 loss, KIAA1549-BRAF fusion

 

  

 

Pyrosequencing

-             MGMT promoter methylation  
MGMT promoter methylation analysis has prognostic and predictive implications for high grade gliomas. The methylation status of four CpG sites within the first exon of the MGMT gene is assessed by bisulfite modification of tumour DNA from fresh or formalin fixed paraffin embedded brain tumour specimens and quantitative pyrosequencing.

MGMT promoter methylation test is one of the MBS rebatable test (MBS item 73373) for eligible patients. 


MGMT promoter methylation status assessed by Pyrosequencing

-              IDH1/2 pyrosequencing
The majority of low-grade diffuse gliomas and secondary glioblastomas have mutations in Isocitrate Dehydrogenase-1 or -2 (IDH-1/2). IDH mutations have been found to carry prognostic significance in both low and high-grade gliomas. We perform pyrosequencing to assess IDH1codon 132 and IDH2 codon 172 mutations on fresh or formalin fixed brain tumour samples.

IDH1/2
 testing is one of the MBS rebatable test (MBS item 73372) for eligible patients. 

-             TERT promoter pyrosequencing
Two high frequency variants, chr5: g1295228 G>A and chr5: g1295250 G>A in TERT promoter positioned respectively 124 and 146 base pairs upstream of the ATG translational start site of TERT, examined by PCR and pyrosequencing. TERT promoter mutations are found in the vast majority of IDH-mutant and 1p/19q co-deleted oligodendrogliomas (present in > 95% of cases) and most IDH-wildtype glioblastomas (present in ~ 80% of cases). TERT promoter mutations are also found in some medulloblastomas (SHH-activated, TP53-wildtype), some meningiomas (usually higher grade meningiomas), some malignant peripheral nerve sheath tumours, and some metastatic melanomas (but not primary CNS melanocytic lesions).

-             H3F3A pyrosequencing
We perform PCR and pyrosequencing to assess codon 27 and codon 34 of H3F3A on fresh or formalin fixed brain tumour samples.H3F3A K27M variant is found in diffuse midline glioma including diffuse pontine gliomas ( e.g. Diffuse midline glioma, H3 K27M-mutant), whereas H3F3A G34R or (rarely) G34V variants are found exclusively in high grade gliomas of the cerebral hemispheres, which are more frequently seen in teenagers and young adults. In paediatrics high-grade gliomas, H3F3A variants grade gliomas, H3F3A variants are associated with variants in the chaperone ATRX and inversely related to IDH variants.