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Molecular Profiling

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Molecular Diagnostics Q&A

Please find below answers to some commonly asked questions relating to the laboratory, the technology and/or the process or ordering tests/receiving reports:

You can also download a document with this information from our Downloads page...


What is personalised medicine and molecular profiling ?

Personalised medicine is an emerging practice of medicine that uses an individual's genetic profile to guide decisions made in regard to the prevention, diagnosis, prognosis or treatment of disease. Personalised medicine for individuals with cancer uses the genetic profile of an individual's tumour to help clinicians select the appropriate medication or therapy and administer it using the proper dose or regimen.  

Molecular profiling is an important facet of personalised medicine, and is the means by which tumours are classified. The genetic profiles are generated using a number of different tests/technologies, the results of which help guide diagnosis and predict response to therapy.

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What is next generation sequencing and why has the laboratory adopted targeted next generation sequencing ?

Next-generation sequencing (NGS), also known as high-throughput or massively parallel sequencing, is the catch-all term used to describe a number of different modern sequencing technologies. These recently developed technologies allow us to sequence large amounts of DNA and RNA much more quickly and cheaply than with the previously used Sanger sequencing approach, and as such have revolutionised the study of genomics and cancer biology. NGS provides a 'genetic survey' of a tissue sample by systematically identifying genetic alterations which - depending on the technology used and methods employed - can include single-nucleotide variants, insertions and deletions (indels), copy number variations, and large genomic rearrangements. Researchers and clinicians now utilise NGS to identify specific changes in DNA by rapidly and simultaneously sequencing multiple gene targets within multiple samples.

 

The Sarah Cannon Molecular Diagnostics laboratory has adopted targeted sequencing on the Ion PGM™ platform. In contrast to whole-genome, exome or whole-transcriptome sequencing, targeted DNA or RNA sequencing focuses the analysis on specific areas of interest. The ability to use targeted sequencing allows NGS to be employed routinely for genetic profiling of tumours in clinical practice and in clinical research. This sequencing approach can help identify rare or novel variants as well as those occurring at low frequency within a sample. When matched with germline DNA, it can also verify the somatic (tumour) nature rare variants across a large number of samples.

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What next generation sequencing platform are you using ?

The laboratory uses the Ion PGM™ (Ion Torrent's Personal Genome Machine) to conduct targeted sequencing of commonly mutated regions (‘hot spots’) in 22 genes commonly implicated in cancer. 

 Ion PGM

What genes are included in the 22-gene panel ?

KRAS, EGFR, BRAF, PIK3CA, AKT1, ERBB2, PTEN, NRAS, STK11, MAP2K1, ALK, DDR2, CTNNB1, MET, TP53, SMAD4, FBXW7, FGFR3, NOTCH1, ERBB4, FGFR1, FGFR2

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Why have these genes been selected ?  And why have you limited the panel to only 22 genes ?

The genes included in the panel are amongst the most common currently actionable 'driver genes' that can be targeted with drugs/ therapeutic agents across an array of cancer sub-types. They have either been selected because the presence or absence of a mutation in one or more of these genes is linked to an authorized medicinal product (e.g., KRAS, NRAS, EGFR, BRAF amongst others) or to an investigational medicinal product currently being (or soon to be) evaluated in clinical trials (e.g., PIK3CA, PTEN, MET amongst others).

Limiting the panel to ‘druggable targets’ is more cost-effective than a more comprehensive panel which may also contain targets that are currently only suitable for research or have limited or no impact on the biology of the disease itself.  For example, in tumours with a high mutational load (non-small cell lung cancer, melanoma, micro satellite unstable colorectal cancer etc), tumours may contain hundreds of thousands of mutations. With no germ line DNA comparator, as is the norm for most commercial tests, expanding the list of genes sequenced increases the cost of providing the assay as well as the risk that genetic events identified will either be passenger events or rare germ line SNPs. As additional genes are identified that are implicated in cancer(s), the existing panel(s) will be modified to include these additional targets.

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What are the advantages of targeted next generation sequencing over single gene tests or multiple sequential single-gene tests ?

The main advantages of multi-gene testing using targeted next generation sequencing are as follows;

  • Tissue: Multiple genes can be tested from a single biopsy within one laboratory analysis. Less DNA/tissue is used in a multi-gene panel as compared to multiple single-gene tests; this is particularly pertinent to disease sub-types where tissue is limited
  • Time: It is quicker to conduct a single multi-gene test than multiple sequential single-gene tests
  • Cost: It is cheaper to conduct a single multi-gene test than multiple sequential single-gene tests
  • Information: NGS provides information regarding the relative frequency with which a mutation is found within a single biopsy; in turn, this information can be used to help inform clinical trial outcome data
  • Flexibility: As new genes are identified that are implicated in cancer, these can be added to NGS panels relatively quickly
  • Options: Multi-gene panel testing is increasingly required to guide patient selection to clinical trials, providing clinicians with more treatment options to offer their patients.

Why do I have to order the 22-gene panel ?  Why can I no longer order single gene tests ?

Increasing evidence suggests that there can be multiple ‘driver genes’ operating within tumour genomes at any one time, therefore a comprehensive analysis of known ‘drivers’ are included in the gene panel to allow clinicians to make more informed and rational decisions about standard of care treatment for their patients or to determine whether individual patients may be suitable for therapies available in one or more clinical trials.  

For example, mutations in KRAS, EGFR, BRAF, PIK3CA, PTEN, AKT1 occur at different frequencies in non-small cell lung cancer (NSCLC): conducting parallel or sequential single-gene tests for each of these genes would exhaust valuable tissue and have cost and/or time implications.

Sarah Cannon Molecular Diagnostics has therefore taken the decision to offer targeted NGS in place of standard single-gene tests in order to support more informed decision-making, and to make better use of valuable resources (tissue, time, money).

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What if I need amplification/ translocation testing ?

The NGS technology we employ with this gene panel is designed to only detect localised mutations, e.g. SNP’s, MNP’s and small scale insertions/deletions. Large scale or whole gene deletions, amplifications, copy number variations, translocations or protein over-expression cannot be determined. If such analyses are required, please continue to order FISH/CISH or immunohistochemistry from Sarah Cannon Molecular Diagnostics as you would normally do. Back to list of questions >>

What types of tissue can be tested ?

The NGS test has been validated for formalin-fixed-paraffin-embedded (FFPE) tissue; this is widely regarded as one of the most difficult sample types to analyse, often yielding only very small quantities of poor quality DNA. Consequently, other less challenging samples such as alcohol fixed cytology specimens, fresh/fresh frozen material, and blood for haematological malignancies can easily be processed as needed.

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Can I use tissue left over from a previous biopsy to look for genetic alterations ?

The NGS test is able to detect mutations in tissue samples which have been stored as formalin-fixed paraffin-embedded (FFPE) material. However, as new driver genetics alterations may have occurred during the course of the disease, the genetic profile of the tumour may have changed since the previous biopsy was taken. Therefore consideration should always be given to whether it is appropriate to use an archived tissue sample or whether a new biopsy should be taken as analysis of this may reveal new genetic events which may influence treatment.

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What are the specimen requirements for next generation sequencing testing ?

Specimen requirements are the same as for single gene tests, which are as follows:

  • Fixed tissue (including FNA, resection specimen, core biopsy etc.): One (1) formalin-fixed paraffin-embedded (FFPE) block from a biopsy or surgical resection should be provided. The combined area of tumour tissue (not total tissue) must be a minimum area of 4mm2.
  • Curls and unstained slides: For large tissue samples (>1.5cm2) 1x10µM section, medium tissue samples (1-1.5cm2) 2x10µM sections, small tissue samples (0.5-1cm2) 3x10µM sections and for multiple small tissue fragments 6-8x10µM sections.

Please provide a stained H&E with the tumour area marked on the slide. Cases which require re-extraction and re-testing due to inadequate indication of tumour load will be charged twice. If on the rare occasion that the H&E slide is not provided, it is essential that all requests include an indication of percentage of tumour DNA content within the sample. Tumour percentage can be estimated as >5%, >25%, >50%, >75%, 100% as often full numeration is not possible. Samples with < 5% tumour content will be returned untested.   

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Can I use your panel to determine whether my patient has a hereditary (germ-line) syndrome ?

No. We do not currently sequence germ-line DNA. At present we only sequence tumour DNA for somatic mutations.

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If there is a gene I am interested in that is not in the panel, how do I go about getting it included ?

Sarah Cannon Molecular Diagnostics welcomes input on the content of its gene panels from clinicians, researchers and companies. If there is a gene in which you are interested that is not currently in the panel, please email the laboratory at info@sarahcannon-md.co.uk clearly defining the gene, mutations of interest and your rationale for their inclusion.

A scientific team comprising representatives from UCL, Sarah Cannon Research Institute, the laboratory and other interested parties will review your request and decide whether to include the gene in the next version of the panel. This team will also determine timelines for its inclusion in the panel.

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What happens if I find unexpected alterations ?

Test results should be discussed through your usual institutional channels e.g., at Multi-Disciplinary Team meetings (MDTs); test results must not be interpreted in isolation and are required to be interpreted in the context of conventional histology reports and the clinical setting, together with established guidelines for drug administration.

Consideration may also be given to whether your patient may be eligible for one or more clinical trials (refer to publicly available resources such as clintrials.gov). Note that although these tests are designed to identify somatic DNA variants, the possibility cannot be excluded that any observed variants may actually be germline in origin (i.e. rare polymorphisms).

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How has the 22-gene next generation sequencing panel been validated ?

The 22 gene NGS sequencing panel being offered by Sarah Cannon Molecular Diagnostics was originally subject to extensive validation by Life Technologies during its initial development, this included a multi-centre consortium study of leading researchers and key opinion leaders using 155 FFPE previously characterised samples.

The technology has since been evaluated within our laboratory using >1,000 samples made up of independently provided controls, rigorously characterised research samples and routine clinical specimens simultaneously co-analysed using current CE-IVD marked single gene assays.

NGS is a developing field and quality assurance is of the highest concern to us: we are therefore constantly auditing our processes and working with a network of accredited clinical diagnostic laboratories across the UK and Ireland to improve the laboratory service.

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How does the sensitivity and specificity of Sarah Cannon Molecular Diagnostics next generation sequencing platforms compare with current standard single-gene tests ?

Although the absolute sensitivity of our targeted NGS approach will vary from sample to sample depending upon variables such as tumour content and the quality/quantity of DNA extracted from any supplied material, overall sensitivity and specificity on any given sample has been demonstrated to be at least as good as traditional single gene tests, and in many cases will be significantly higher.

Furthermore the assay is not constrained by the detection of only a limited subset of the most common variants as in the case of most single gene tests. Rare and novels variants can be detected with the same sensitivity as previously well characterised ones.

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How is the mutation test reported ?

Any coding variants identified will be reported in the format 'Gene name, protein change, cDNA change, COSMIC reference'. These will be listed in the first section of the supplied report which is divided into two sub-sections, the first details any variants identified in genes that we have previously offered as single gene assays (BRAF, EGFR, KRAS, NRAS) the second details all other identified variants. 

The remainder of the report summarises those variants which were not detected, and details any genomic regions where sequence data may have been missing or suboptimal for a rigorous analysis. Please note that no interpretive comments are provided with regards to individual variants.

This report can be sent by facsimile or email if required.

An example of the NGS report can be downloaded here. Please refer to the assay guidelines on page 3 for explanatory help.

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Does the laboratory participate in any quality control networks ?

The laboratory participates in the NEQAS schemes for single gene testing of BRAF, KRAS and EGFR and plans to participate in the new NEQAS scheme for somatic mutation testing by NGS in the future. In the meantime, in line with accreditation guidelines, the laboratory is working with other laboratories to set up an informal EQA scheme with a network of other laboratories offering similar tests utilising the same technology. 

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How long does the NGS test take ?

Provided that sample material and accompanying paperwork meets minimum acceptance criteria, reports will normally be available within 8-10 working days of receipt. Samples with < 5% tumour content will be returned untested and samples provided with incomplete details/paperwork or requiring pathologist review will not be processed until this is complete. In the unlikely event of analysis failure on a specific sample, unfortunately the only option is to repeat the entire process which may add a further 4-5 days to the total turnaround.

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Can I expedite urgent tests ?

Unfortunately, the full NGS pipeline necessitates a stepped multiday workflow, no stage of which can be by-passed. Every effort will be taken to ensure that specimens enter the pipeline as soon as possible on receipt, and any cases identified as urgent will be analysed and reported as a priority upon completion of laboratory phase of the analysis. Other than this, there is no technically possible way to expedite testing irrespective of clinical urgency.

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How do I order the test ?

Request forms are available at Sarah Cannon Molecular Diagnostics

 

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If you have additional questions, please do not hesitate to contact the Molecular Profiling Laboratory:

Telephone: +44 (0)20 3794 1920

Email:  info@sarahcannon-md.co.uk

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