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.
The Anaplastic Lymphoma Kinase (ALK) gene encodes a tyrosine kinase normally expressed in neuronal cells. The ALK gene can, by deletion or translocation, form a fusion protein which is abnormally expressed in approximately 2 to 5% of NSCLC patients. The presence of this ALK gene rearrangement predicts response to ALK inhibitor therapy. Response to ALK inhibitors is also predicted by the presence of translocations involving the structurally related ROS1 proto-oncogene. ALK and ROS1 rearrangements are typically mutually exclusive of EGFR and KRAS mutations. KRAS mutations are frequently found in lung adenocarcinomas of patients with a smoking history (approximately 40% incidence).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 prevents T-cell activation and contributes to the evasion of tumour cells from immune recognition. Immune checkpoint inhibitors such as nivolumab and pembrolizumab are monoclonal antibody therapies that target PD-1 on the patient’s T-cells, thereby blocking PD-1/PD-L1 interaction and preventing tumour cells from inactivating the local cytotoxic T-cell response.After DNA isolation, Next generation sequencing (NGS) is performed to detect EGFR and KRAS mutations, and immunohistochemistry (IHC) or RNA NGS fusion testing is used to assess expression of the ALK and ROS1 proteins. Tumours positive for ALK or ROS1 by IHC, or those showing an equivocal pattern of staining, are further tested by fluorescence in-situ hybridisation (FISH) to confirm the presence of a gene rearrangement. Upregulation of PD-L1 expression in tumour cells is demonstrated by IHC using the Dako PD-L1 IHC 22C3 pharmDx kit.EGFR mutation testing for stratification of first line treatment is usually performed using DNA isolated from FFPE tissue samples. 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.Associated tests
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
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.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:
Accurate classification of brain tumours is essential for selection of appropriate treatment modalities. Detection of mutations in IDH1 and IDH2 assists the differential diagnosis of gliomas and provides key diagnostic and prognostic information: IDH mutation status is a definitive marker of secondary glioblastoma; astrocytomas and oligodendrogliomas can contain mutations in IDH1 or IDH2, whereas other CNS tumours usually do not contain these mutations.Glioblastoma is the most common and most aggressive form of brain cancer. Patients with high grade glioblastoma may receive alkylating chemotherapy with temozolomide as first-line treatment. Methylation of the promoter of the MGMT gene, which encodes a DNA repair enzyme, predicts for response to chemotherapy with alkylating agents.Associated tests
Trastuzumab, in combination with cisplatin and capecitabine or 5-fluorouracil, is an option for the treatment of people with human epidermal growth factor receptor 2 (HER2 [ERBB2]) amplified metastatic adenocarcinoma of the stomach or gastro-oesophageal junction who have not received prior treatment for their metastatic disease. Response is predicted by establishing HER2 protein overexpression by immunohistochemistry in combination with direct demonstration of gene amplification by fluorescent in-situ hybridisation (FISH).Associated Tests
Tumours of mesenchymal origin represent a complex group that includes bone and soft tissue sarcomas, haemangiomas, schwannomas and gastrointestinal stromal tumour (GIST).KIT and PDGFRA mutations are a characteristic finding in GIST that aids histopathological diagnosis and help predict response to therapy.In-situ hybridisation studies are routinely used as an adjunct to morphological assessment in the classification of soft tissue and bone tumours, many of which are characterised by distinctive cytogenetic abnormalities, and in particular for cases that present a diagnostic challenge. Currently in routine use we offer:DDIT3 (breakapart) – myxoid liposarcomaEWSR1 (breakapart) – Ewing’s sarcoma, primitive neuroectodermal tumour (PNET), myxoid chondrosarcomaFOXO1 (breakapart) – alveolar rhabdomyosarcomaFUS (breakapart) – low-grade fibromyxoid sarcoma, myxoid liposarcoma and angiomatoid fibrous histiocytomaMDM2 (amplification) – liposarcomaSS18 (breakapart) – synovial sarcomaUSP6 (breakapart) – aneurysmal bone cyst, nodular fasciitisAssociated Tests
Thyroid Cancer
KRAS, NRAS, HRAS (collectively known as “RAS”), BRAF and TERT promoter mutation analysis is offered as an aid for the pathological diagnosis and subsequent management of patients with suspected thyroid cancer. Testing is especially valuable for patients with indeterminate cytology, where the detection of a BRAF, RAS or TERT promotor mutation would strongly favour the diagnosis of thyroid cancer. BRAF and TERT mutations either individually or combined are 100% predictive of malignancy. RAS mutations are found in both benign and malignant disease, but some RAS mutations are associated with malignancy.
BRAF, RAS or TERT promotor mutation status allow prognostication and stratification of risk, in both cytology samples and surgical resections. Such information is of crucial importance to identify patients who will benefit from more aggressive treatment options.
Associated tests
HRAS mutation analysis
Endometrial cancer
Emerging evidence has revealed that molecular profiling of endometrial cancer can provide robust prognostic and predictive information that can help to escalate or de-escalate adjuvant therapy.
The presence of POLE mutations tends to be associated with a favourable prognosis while mismatch repair (MMR) deficient endometrial cancers carry an intermediate prognosis and those with TP53 mutations carry a poor prognosis. Endometrial cancers with both POLE and TP53 mutations or both MMR deficiency and TP53 mutations carry a prognosis similar to POLE mutant and MMR deficient tumours respectively.
Associated tests
Granulosa Cell (Ovarian) Tumours
Adult granulosa cell tumours (AGCTs) are low-grade malignant sex cord-stromal tumours of the ovary. They are uncommon, accounting for less than 5% of malignant ovarian tumours. Their growth is slow and indolent, but local recurrence or metastasis occurs in up to 20-30% of cases,
The FOXL2 c.402C>G p.(Cys134Trp) variant is detected in ~95% of AGCTs and not detected in benign cellular fibromas (CFs). Therefore, the presence or absence of a FOXL2 c.402C>G p.(Cys134Trp) aids clinicians with the accurate disease classification.
Associated tests
Pharmacogenomics
Pharmacogenomics refers to the study of the way a patient's genome affects their response to drugs. By identification of certain germline variants in genes involved in drug metabolism, it is possible to predict response, and in particular adverse reactions, to a range of common cancer therapies. Dosing decisions can then be tailored for the individual, and potantially life-treatening toxicity avoided.
Germline variants in the dihydropyrimidine dehydrogenase (DPYD) gene can confer an increased risk of severe toxicity when a patient is treated with the fluoropyrimidines, capecitabine or 5-fluorouracil. DPYD testing is performed on DNA extracted from peripheral blood samples.
Associated Tests
Other Services
The Molecular Pathology Laboratory is equipped to offer expert advice as well as mutation analysis, DNA extraction and/or sequencing services for diagnostic and research purposes. Please contact the Molecular Pathology team for further information.We also offer a robust patient identification assay to aid resolution of potential specimen mix-ups arising within the Department of Laboratory Medicine. This test is accredited to ISO 15189.