Introduction

Chimeric gene fusions involving the ALK (anaplastic lymphoma kinase) gene are now recognised as oncogenic driver mutations in about 5% of all NSCLC. The most common genomic rearrangement involving the ALK gene is an inv(2)(p21p23), resulting in the creation of an EML4-ALK chimeric gene, although over 20 other partner genes have been described. Tumours with ALK genomic rearrangements are now considered as a new molecular subgroup of lung carcinomas. Tumours with ALK rearrangements show excellent response rates to inhibitors of ALK tyrosine kinase activity e.g. Crizotinib. Testing for ALK rearrangements can be by several methods e.g. ALK1 IHC, FISH analysis or reverse transcriptase PCR. ALK FISH analysis is regarded as the 'gold standard' method.

Referral information

Biopsies, resections or cytology samples from patients where ALK rearrangement status is required to guide future treatment. If required, clinical advice on suitability of the patient for ALK testing is available from Dr F Blackhall (Fiona.Blackhall@christie.nhs.uk). We require formalin fixed paraffin embedded (FFPE) material that has been reviewed by a histo/cyto-pathologist to identify areas containing tumour cells and determine suitability for testing. A minimum of 2 x 4µM sections are required. Sections should be cut using a microtome and floated on the surface of a purified waterbath at 40+/-2°C. The sections should then be mounted onto positively charged glass slides and air dried. An H&E stained slide clearly marked with the areas of tumour to be examined should accompany all sections for FISH testing. Please contact the laboratory for guidance if you are unsure whether a sample is suitable. ALK1 IHC pre-screening may be useful in selecting patient samples likely to have ALK rearrangement, please contact the laboratory for further information. The laboratory can also offer testing for the NSCLC-associated ROS1 rearrangement. Please contact the laboratory for details.

View full test details for ALK (FISH) testing in NSCLC

Introduction

Neurofibromatosis is an autosomal dominant tumour predisposition syndrome characterized by café au lait spots and fibromatous tumors of the skin. A subset of patients present with NF1 like symptoms which may be due to segmental NF1 mosaicism or to mutations in the SPRED1 gene. ≈8% of patients with café au lait spots only have mutations in SPRED1.

Referral information

The service is NCG funded and is available for patients satisfying clinical criteria of segmental NF1 Samples will only be accepted after clinical review by Dr Burkitt-Wright or Prof Evans of the Dept of Medical Genetics, St Mary’s Hospital Manchester and a clinical proforma must be completed to be sent into the lab upon referral.

View full test details for Atypical Neurofibromatosis Type I

Introduction

BRAF is a proto-oncogene mutated in many types of cancers. The majority of BRAF mutations described occur at codon 600. Codon 600 mutations are present in between 40-60% of malignant melanomas. Melanomas with a mutation at codon 600 of BRAF show a higher likelihood of response to treatment with BRAF inhibitors e.g. Vemurafenib. Somatic mutations in NRAS codons 12, 13 or 61 have been found in ~13–25% of all malignant melanomas and their presence or absence can be used to guide appropriate therapy. Somatic mutations in KIT have been found in 2–8% of all malignant melanoma.

Referral information

Referrals are from oncologists and samples are received via pathologists. Samples should have undergone pathology review to ensure that the sample submitted for analysis is of appropriate pathology and the tumour cell content is sufficient to permit mutation detection. Biopsies, resections or cytology samples from newly diagnosed patients with melanoma where BRAF, NRAS, KIT status is required to guide future treatment. Formalin fixed paraffin embedded (FFPE) tissue is accepted, however the tissue must be sent as a minimum of 5 x 5µM thick sections sealed in a clean 1.5mL Eppendorf tube. A minimum of 20% tumour tissue in the submitted sample is required for optimum mutation detection in BRAF, NRAS and 30% for KIT (see sample requirements).

View full test details for BRAF, NRAS, KIT Mutation Testing in Melanoma Samples

Introduction

The management of families at high risk of having an inherited BRCA1/2 mutation where there is no living affected relative and/or no DNA sample is available, may benefit from BRCA1/2 mutation testing of archive pathology material. The test has been designed to perform on DNA extracted from typical formalin fixed paraffin embedded (FFPE) tissue specimens. BRCA1/2 mutation screening of FFPE tissue can identify germline pathogenic BRCA1/2 mutations in deceased relatives and a negative result can also reduce the likelihood that a pathogenic BRCA1/2 mutation is present in the family.

Referral information

We require a minimum 5x5µM thick unstained sections preferably from a pathology specimen comprising normal (non-cancerous) tissue which must have been reviewed by a histopathologist clearly stating the proportion (if any) of neoplastic cells in the sample. The sample must be accompanied by a completed test request form. Referrals will be accepted from Clinical Geneticists or other appropriate health care professionals. Consent to obtain the material and undertake the testing must accompany the test request.

View full test details for BRCA1/2 mutation screening in FFPE samples to assist in the management of familial breast/ovarian cancer

Introduction

The WHO 2016 guidelines for classification of tumours of the central nervous system integrates molecular genomic information with histological analysis. The central nervous system tumour service offers a range of tests for the differential diagnosis of CNS tumours, provides prognostic information, and may aid in treatment decisions or clinical trial recruitment.

Referral information

We require a minimum 4x5µM thick unstained sections from a pathology specimen, either slide mounted or as scrolls (scrolls are required for KIAA1549-BRAF fusion testing), which must have been reviewed by a histopathologist and be accompanied by a completed test request form, clearly stating the proportion of neoplastic cells in the block. For optimum detection of somatic mutations >20% neoplastic cell content is required (>30% for TERT promoter mutation testing). Referrals will be accepted from oncologists and pathologists. If MGMT hypermethylation or KIAA1549-BRAF fusion testing is requested in addition to another test, we require an additional 4x5µM sections.

View full test details for Central Nervous System Tumours

Introduction

EGFR is a protein found at high levels in many types of cancers, particularly non-small cell lung cancer (NSCLC). Activating mutations in the EGFR gene are present in a proportion of NSCLC tumours. Tumours with mutated EGFR show a higher likelihood of response to treatment with EGFR tyrosine kinase inhibitor drugs e.g. Iressa (Gefitinib).

Referral information

Biopsies, resections or cytology samples from patients with NSCLC that have not undergone chemotherapy. Formalin fixed paraffin embedded (FFPE) tissue is preferred. We recommend 10 x 10µM thick unstained, unmounted sections sealed in a clean 1.5mL Eppendorf tube. The tissue sample should have been reviewed by a histopathologist to determine suitability for testing and estimate tumour cell content. A 30% ratio of tumour to normal tissue in the submitted sample is required for optimum mutation detection. Other samples may be acceptable and macrodissection can be considered for samples with low tumour cell content, however, please contact the laboratory for guidance first.

View full test details for EGFR Mutation Testing in NSCLC (Adenocarcinoma) Samples

Introduction

EGFR-activating mutations are detected in ~11% of NSCLC (adenocarcinoma) UK patients and these patients show a higher likelihood of response to treatment with EGFR tyrosine kinase inhibitors. Adequate biopsy material is not always available as patients may be too unwell or the neoplastic cell content is too low for EGFR mutation analysis. DNA circulating in plasma from blood can be used as an alternative where a biopsy is inadequate as DNA is shed from tumour tissue at increased level in patients with active or advanced disease. Blood samples must reach the laboratory within 24hrs of sampling (ideally within 6hrs). Where this is not possible please contact the laboratory for advice.

Referral information

Blood samples from patients with a diagnosis of NSCLC where biopsy material is not available or inadequate (e.g. too little material, neoplastic cell content <10%). Alternatively blood samples from patients with biopsy proven EGFR mutated disease who have progressed following EGFR-TKI therapy where the results are being used to monitor progression, molecular resistance or enrolment on clinical trials.

View full test details for EGFR Mutation Testing in plasma (cfDNA)

Introduction

Heterozygous mutations in APC cause familial adenomatous polyposis (FAP).  Affected individuals usually develop hundreds to thousands of adenomatous polyps of the colon and rectum, a small proportion of which will progress to colorectal carcinoma if not surgically treated.  MUTYH-associated polyposis (MAP) is an autosomal recessive disorder caused by bi-allelic pathogenic mutations in MUTYH.  Typically associated with tens to a few hundred colonic adenomatous polyps, MAP also greatly increases lifetime risk of colorectal cancer.

Referral information

We accept referrals for mutation scanning in APC and MUTYH from patients affected by and with a family history of bowel cancer. Patients must have been seen by a Clinical Geneticist and their risk of FAP or MAP assessed.  If a pathogenic mutation is found we can offer follow-up testing to at-risk family members.

View full test details for Familial Adenomatous Polyposis and MUTYH-Associated Polyposis

Introduction

Mutations in the germ-line of predisposition genes account for approximately 5-10% of women affected with breast cancer. Two genes BRCA1 and 2 account for the majority of familial cases.

Referral information

We accept referrals for mutation scanning in BRCA1 and BRCA2 from patients affected by and with a family history of breast cancer. Patients must have been seen by a Clinical Geneticist and must meet the criteria for genetic testing indicated in the guidelines issued by NICE. Screening for three common mutations in the Ashkenazi Jewish population and in the Polish population can also be offered if appropriate. If a mutation has been identified in the family, predictive/confirmatory testing can be offered to at-risk family members.

View full test details for Familial Breast Cancer

Introduction

Gastro-intestinal stromal tumours (GISTS) are a rare form of gastrointestinal neoplasm, however they are the most common mesenchymal tumour of the gastrointestinal tract.  Approximately 700 new cases are diagnosed each year in the UK.  Diagnosis is mainly by clinical features and standard histopathology including immunostaining.

A majority of GISTs have been found to have activating mutations in one of two tyrosine kinase genes KIT and PDGFRA.  The majority ~65% have acquired activating mutations in KIT whereas ~3% have acquired activating mutations in PDGFRA.

Imatinib is useful in the clinical management of patients with GIST, with the majority of GISTs showing a good response to imatinib therapy.  Imatinib is only approved for use in GIST where the diagnosis has been confirmed either by KIT immunostaining or positive mutation analysis.

Referral information

Formalin fixed paraffin embedded (FFPE) tissue is preferred. We recommend 10 x 10µM thick unstained, unmounted sections sealed in a clean 1.5mL Eppendorf tube. The tissue sample should have been reviewed by a histopathologist to determine suitability for testing and estimate tumour cell content. A 20% or greater ratio of tumour to normal tissue in the submitted sample is required for optimum mutation detection. Other samples may be acceptable and macrodissection can be considered for samples with low tumour cell content, however, please contact the laboratory for guidance first.

View full test details for Gastrointestinal Stromal Tumour

Introduction

KRAS, NRAS and BRAF are oncogenes frequently somatically mutated in colorectal tumours. Somatic KRAS, NRAS and BRAF mutations result in a specific gain of function and almost exclusively affect codons 12, 13 and 61 of the KRAS and NRAS genes and codon 600 of BRAF. As KRAS, NRAS and BRAF lie downstream of EGFR in the EGFR signalling pathway, presence of an activating KRAS, NRAS or BRAF mutation in a colorectal tumour predicts a likely poor response to EGFR inhibiting therapy. Frequency of KRAS, NRAS, BRAF mutations in colorectal cancer (CRC) samples is approximately 36–40%, 1-6% and 8–15% respectively.

Referral information

Colorectal cancer samples from recently diagnosed patients.  Referrals are from Oncologists and samples are received via pathologists. Samples should have undergone pathology review to ensure that the sample submitted for analysis is of appropriate pathology and the tumour cell content is sufficient to permit mutation detection. Formalin fixed paraffin embedded (FFPE) tissue is accepted, however the tissue must be sent as a minimum of 5 x 5µM thick sections sealed in a clean 1.5mL Eppendorf tube. A minimum of 20% tumour tissue in the submitted sample is required for optimum mutation detection (see sample requirements).

View full test details for KRAS, NRAS and BRAF Mutation Testing in Colorectal Tumour Samples

Introduction

Legius syndrome is an autosomal dominant disorder that shows some similarities to neurofibromatosis type I (NF1; 162200), which is caused by mutation in the neurofibromin gene (613113); however, Legius syndrome is less severe. Individuals with Legius syndrome typically have multiple cafe-au-lait spots, sometimes associated with skin fold freckling, variable dysmorphic features such as hypertelorism or macrocephaly, lipomas, and mild learning disabilities or attention problems. Legius syndrome is not associated with neurofibromas, optic gliomas, Lisch nodules, or tumour predisposition. ≈8% of patients with café au lait spots only have mutations in SPRED1.

Referral information

The service is NCG funded and is available for patients satisfying clinical criteria of segmental NF1. Samples will only be accepted after clinical review by Dr Emma Burkitt-Wright or Prof Evans of the Dept of Medical Genetics, St Mary’s Hospital Manchester and a proforma must be completed.

View full test details for Legius Syndrome

Introduction

Lynch syndrome is an autosomal dominant disorder with a high penetrance rate and accounts for 3%-4% of all colorectal cancer cases. Allelic heterogeneity and phenocopies occur. At least six mismatch repair genes may be involved in Lynch syndrome pathology. Germ line mutations in the MLH1 and MSH2 genes have been estimated to be responsible for approximately 60-70% of HNPCC cases.

Referral information

We accept referrals for mutation scanning in MLH1, MSH2 and MSH6 from patients affected by and with a family history of bowel cancer. Patients must have been seen by a Clinical Geneticist and their risk of Lynch syndrome assessed against the Amsterdam criteria.  If a pathogenic mutation is found we can offer follow-up testing to at-risk family members.

View full test details for Lynch Syndrome (Previous known as Hereditary Non-Polyposis Colon Cancer, HNPCC)

Introduction

MLH1 loss determined by IHC is observed in dMMR tumours from both Lynch syndrome patients but also a significant minority of sporadic tumours. MLH1 promoter hypermethylation is a common somatic epimutation in sporadic tumours yet is rare in Lynch syndrome tumours where the ‘first hit’ is inherited. MLH1 promoter hypermethylation can be used to stratify Lynch syndrome risks in patients, particularly those which meet revised Bethesda criteria.

Referral information

We accept biopsies or resection samples from patients with a possible diagnosis of Lynch syndrome. Formalin-fixed paraffin-embedded (FFPE) material should have been reviewed by a pathologist to determine suitability for testing and an estimate of tumour cell content is required. We require either 5 x 5µm unstained, slide-mounted sections or unmounted sections from tissue blocks containing a) tumour tissue and b) normal tissue.  A 30% ratio of neoplastic to normal cells in the submitted pathology sample is required for reliable MLH1 promoter methylation assessment. Samples with lower tumour cell content may be acceptable but wherever possible should be accompanied by a representative H&E stained slide with areas of tumour marked to allow for macrodissection. Please contact the laboratory for guidance if you are unsure whether a sample is suitable. Lymphocyte samples are accepted in order to test for rare cases of Lynch syndrome due to constitutional MLH1 promoter hypermethylation.

View full test details for MLH1 Promoter Hypermethylation

Introduction

MSI is a change in length of a microsatellite allele due to insertion or deletion of repeating units during DNA replication and failure of the DNA mismatch repair system to correct these errors.  Samples with instability in two or more markers are defined as MSI-High (MSI-H) whereas those with one unstable marker are designated as MSI-Low (MSI-L).  Samples with no detectable alterations are MSI-stable (MSS).

Referral information

We accept biopsies or resection samples from patients with a possible diagnosis of Lynch syndrome.   MSI testing is also available in the context of contributing to decision-making regarding chemotherapy administration. Formalin-fixed paraffin-embedded (FFPE) material should have been reviewed by a pathologist to determine suitability for testing and an estimate of tumour cell content is required. We require either 5 x 5µm unstained, slide-mounted sections or unmounted sections from tissue blocks containing a) tumour tissue and b) normal tissue.  A 20% ratio of neoplastic to normal cells in the submitted pathology sample is required for reliable MSI assessment. Samples with lower tumour cell content may be acceptable but wherever possible should be accompanied by a representative H&E stained slide with areas of tumour marked to allow for macrodissection. Please contact the laboratory for guidance if you are unsure whether a sample is suitable.

View full test details for Microsatellite Instability (MSI)

Introduction

NF2 is an autosomal dominant cancer syndrome affecting approximately 1 in 40,000 individuals and is characterized by the formation of multiple benign nervous system tumours, most commonly bilateral schwannomas of the vestibular nerve. NF2 is caused by mutations in the schwannomin/merlin tumour suppressor gene located on chromosome 22q12.2.

Referral information

Mutation screening in patients with clinically confirmed, or a differential diagnosis, of NF2. Pre-symptomatic testing of relatives of an index case with previously identified mutation or by linkage in families with suitable pedigree structure (samples from at least two affected first degree relatives).

View full test details for Neurofibromatosis type 2 (NF2)

Introduction

The PIK3CA gene encodes a catalytic subunit of phosphatidylinositol 3-kinase (PI3K). Activation of the PI3K-AKT pathway is believed to play an oncogenic role in many cancer types. Somatic activating mutations in PIK3CA have been described in a wide range of tumour types including breast, ovarian and colorectal cancers. Mutations in PIK3CA are predominantly found at three functionally relevant hotspots located at codons 542, 545 & 1047.

Referral information

Biopsies, resections or cytology samples from patients where PIK3CA status is required for enrolment on clinical trials or to guide future treatment. We require formalin fixed paraffin embedded (FFPE) material that has been reviewed by a histo/cyto-pathologist to estimate tumour cell content and determine suitability for testing. A 20% ratio of tumour to normal cells in the submitted sample is needed for optimum mutation detection. For samples meeting this requirement send 5 x 10µM thick unstained, rolled sections in a clean container. Samples with lower tumour cell content may be acceptable but should be sent as unstained 5µM thick slide mounted sections (without coverslips) accompanied by a representative H&E stained slide with the area(s) of tumour marked to allow for macrodissection. Please contact the laboratory for guidance if you are unsure whether a sample is suitable.

View full test details for PIK3CA Mutation Testing

Introduction

Schwannomatosis is a tumour predisposition syndrome showing autosomal dominant inheritance. There is clinical overlap between schwannomatosis and NF2. Diagnostic criteria for schwannomatosis include 2 or more pathologically proved schwannomas and lack of radiographic evidence of vestibular nerve tumor at age more than 18 years (Jacoby et al 1997). Rhabdoid tumors are a highly malignant group of neoplasms that usually occur in children less than 2 years of age.

Referral information

Lymphocyte DNA from individuals satisfying clinical diagnostic criteria for Schwannomatosis (e.g. 2 or more schwannomas no vestibular schwannomas) where NF2 mutation screening has proved negative. All schwannomatosis referrals should be accompanied by a completed clinical questionnaire to assist with the audit of the service (available from www.mangen.org.uk). Lymphocyte DNA from individuals diagnosed with Atypical Familial Rhabdoid Tumour.

View full test details for Schwannomatosis -SMARCB1/LZTR1; Atypical Familial Rhabdoid Tumour - SMARCB1.

Introduction

Oncogene driver mutations have been identified across a wide range of tumours and therapies are increasingly being adopted into standard clinical practice whose clinical effectiveness is dependent on the presence or absence of oncogene mutations. Panel testing for a wide range of oncogene mutations uses limited pathology material effectively, reduces overall test turn-round and is more cost effective than serial testing. Panel testing can also be used to pre-screen patients for clinical trial eligibility. This test identifies oncogene driver mutations (single nucleotide changes and insertion/deletions <40bp) across 24 clinically relevant oncogenes.

Referral information

We require a minimum 5x5uM thick unstained sections from a pathology specimen which must have been reviewed by a histopathologist and be accompanied by a completed test request form, clearly stating the proportion of neoplastic cells in the block. For optimum detection of somatic mutations >20% neoplastic cell content is required.

Referrals will be accepted from oncologists, pathologists and MDTs managing  cancer patients with a range of solid tumours including cancers of unknown primary.

View full test details for Somatic Cancer Next Generation Sequencing (NGS) Mutation Panel

Introduction

Patients with platinum sensitive relapsed ovarian cancer and germline or somatic mutations in BRCA1/2 may benefit from treatment with the poly ADP ribose polymerase (PARP) inhibitor Lynparza (Olaparib). BRCA1/2 mutation screening in FFPE tissue can identify patients with somatic BRCA1/2 mutations who may benefit from targeted therapy. The test has been designed to perform on DNA extracted from formalin fixed paraffin embedded (FFPE) tissue specimens and detect somatic mutations in samples with neoplastic cell content >20%.

Referral information

We require a minimum 5x5µM thick unstained sections from a pathology specimen which must have been reviewed by a histopathologist and be accompanied by a completed test request form, clearly stating the proportion of neoplastic cells in the block. For optimum detection of somatic mutations >20% neoplastic cell content is required. Referrals will be accepted from oncologists, pathologists, geneticists and MDTs managing patients with platinum sensitive relapsed ovarian cancer.

View full test details for Tumour BRCA1/2 mutation testing in Platinum sensitive relapsed ovarian cancer patients

Introduction

UDP-glucuronosyltransferase-1A1 or UGT1A1 is an enzyme encoded by the UGT1A1 gene. UGT1A1 is involved in metabolism of several drugs. The promoter of the UGT1A1 gene contains a common variant in the region of the TATA box promoter element. The most common variant A(TA-6)TAA accounts for ~50% of alleles whereas the UGT1A1*28 variant which has an extra TA dinucleotide A(TA-7)TAA can account for up to 40% of alleles. UGT1A1*28 is associated with lower expression levels of UGT1A1. Individuals who are homozygous for the UGT1A1*28 allele (~15% of the population) show reduced levels of glucoronidation by UGT1A1 and are at raised risk of a mild syndrome of hyperbilirubinaemia under conditions of stress (Gilbert syndrome). There is evidence that UGT1A1*28 homozygotes are at raised risk of adverse reactions to drugs that are metabolised by this pathway including irinotecan. UGT1A1*28 status can be used to guide dosage for irinotecan to minimise adverse reaction.

Referral information

Please contact the laboratory to discuss your requirements.

View full test details for UGT1A1-28 Genotyping