Testing

Following a request for testing, pathology reports are reviewed and the most appropriate specimen selected.  Haematoxylin and eosin (H&E) stained sections are assessed to identify a tumour area suitable for molecular testing based on the proportion of neoplastic cells, the limits of detection of each assay and the size of the available sample.  Where necessary, tissue sections may be macrodissected to increase the proportion of tumour DNA in the sample; this is an essential prerequisite for molecular pathology testing as failure to enrich tumour DNA can result in false negative results.

 

DNA is isolated from the identified areas using the Qiagen QIAamp DNA FFPE Tissue Kit.

 

Somatic mutations in exons 18 to 21 of the Epidermal growth factor receptor (EGFR) gene are used to predict response to EGFR tyrosine kinase inhibitors (TKIs).  EGFR TKIs are currently used as first line treatment for patients with advanced metastatic non-small cell lung cancer (NSCLC) and are associated with an improved prognosis compared to chemotherapy. EGFR mutations in FFPE-tissue samples are assessed by Next Generation sequencing (NGS). Testing can also be performed using a real-time PCR method on cell-free tumour DNA (cfDNA) isolated from peripheral blood.  cfDNA analysis is most applicable for confirmation where the emergence of mutations conferring resistance to EGFR TKI therapy is suspected.

The presence of a Kirsten rat sarcoma virus (KRAS) G12C mutation, specifically, predicts response to KRAS G12C targeted inhibitors. In NSCLC samples other hot-spot KRAS mutations have been found to be largely mutually exclusive with both EGFR mutations and ALK or ROS1 gene rearrangements, but have no relevance to treatment selection at this time.  KRAS mutations are detected by NGS.

Fusion proteins resulting from gene-rearrangement of the Anaplastic Lymphoma Kinase (ALK) gene, ROS1 proto-oncogene, RET proto-oncogene, or NTRK1, NTRK2 or NTRK3 genes predict response to targeted TKIs. Immunohistochemistry (IHC) is used to assess aberrant expression of the ALK and ROS1 proteins that could indicate gene-rearrangement, which is subsequently confirmed by fluorescence in-situ hybridisation (FISH) and/or RNA fusion NGS. RET gene-arrangement is assessed by FISH and/or RNA fusion NGS. The presence of NTRK1, NTRK2 or NTRK3 gene fusions is assessed by RNA fusion NGS. 

 

Alterations that lead to loss of exon 14 of the MET gene (MET exon 14 skipping) predict response to selective MET TKIs. MET exon 14 skipping may be assessed by RNA fusion NGS.

 

Programmed death 1 (PD-1) is an immune inhibitory receptor that is widely expressed by cells of the immune system, especially cytotoxic T lymphocytes.  Upregulation of the ligand PD-L1 by tumour cells prevents T-cell activation and contributes to the evasion of the tumour cells from immune recognition.  Immune checkpoint inhibitors that target PD-1 block the PD-1/PD-L1 interaction and prevent tumour cells from inactivating the local cytotoxic T-cell response. Upregulation of PD-L1 expression in tumour cells is assessed by IHC.

 

Note RNA fusion NGS is currently performed by North East Scotland Genetics Service, NHS Grampian.

 

Associated tests

EGFR mutation analysis (FFPE)

EGFR mutation analysis (blood)

ALK gene rearrangement analysis

ROS1 gene rearrangement analysis

RET gene rearrangement analysis

KRAS mutation analysis

PD-L1 immunohistochemistry

 

Anti-EGFR monoclonal antibody therapy is currently used in combination with chemotherapy in metastatic colorectal tumours that carry no mutations in exons 2, 3 or 4 the KRAS or NRAS genes.  The presence of a RAS mutation predicts for a lack of response to therapy.  The presence of a BRAF mutation (in the absence of microsatellite instability) is associated with poorer overall prognosis in colorectal cancer.

 

The Laboratory also performs routine screening for Lynch Syndrome, a form of predisposition to development of colorectal cancer caused by hereditary defect in the DNA mismatch repair (MMR) pathway.

Associated Tests

KRAS mutation analysis

NRAS mutation analysis

BRAF mutation analysis

Mismatch repair testing

 

Melanoma is a form of skin cancer originating in the pigment-producing melanocytes of the epidermal basal layer.  Melanoma patients with tumours that harbour mutations in codon 600 of the BRAF gene are likely to respond to BRAF inhibitor therapy and/or MEK inhibitor therapy.  Some melanomas also harbour mutations in NRAS and KIT

 

Assessment of GNAQ and GNA11 may also be performed upon request.

 

Melanin is a known inhibitor of PCR reactions which may lead to a higher than expected test failure rate in melanoma specimens (see factors known to affect Molecular Pathology testing). 

Associated tests:

BRAF mutation analysis

NRAS mutation analysis

KIT mutation analysis