How to get the best results with Next-Generation Sequencing (NGS)

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There are two main reasons why NGS can fail

  1. There is not enough tumour tissue in the sample (Quantity Not Sufficient);
  2. The DNA in the sample is compromised (Quality Not Sufficient).

How to avoid a ‘Quantity Not Sufficient’ sample

  • A total of 50 mm2 of tumour tissue is typically sufficient, as it contains an amount of at least 50 ng of DNA – the minimum amount needed for NGS.
  • The type of biopsy used affects the success rates of obtaining sufficient tissue for NGS. Core biopsies (99.3% success rate) and excisional biopsies (96.1%) have been shown to more likely be successful than endoscopic biopsies (63.6%) or fine needle aspirate biopsies (39.4%).1

How to avoid a ‘Quality Not Sufficient’ sample

  • It is critical that the tissue specimen is immersed in the fixative solution as soon as possible after it has been taken, and that it is fixed for an appropriate duration. Caris recommends a fixation duration of 12-24 hours.
  • The fixative solution penetrates the specimen at about 1 mm/hour. Tissue that is thicker than 4 mm should be cut before fixing to ensure an even penetration.
  • The fixative solution should only contain formalin and phosphate buffer. Other additives used in the past, such as an acidic buffer, may inhibit NGS.
  • Formalin solution should be used within 24 hours after dilution to 4% to ensure a stable concentration.
  • Avoid using paraffin blocks older than 6 months as a long storage time of the tissue block in paraffin increases the likelihood of failure.

Ensuring an appropriate specimen preparation 

  • Please refer to the ‘Specimen Preparation Instructions’ document for recommendations and requirements on collecting a sufficient sample, verifying tumour content and preparing a Formalin Fixed Paraffin Embedded (FFPE) specimen.
  • Caris generally requests more slides than may ultimately be used to ensure a full NGS analysis can be performed for every patient.
  • Please be aware of profiling services who pride themselves on using less tissue/DNA for NGS. A consortium of international oncologists has suggested that some platforms should be avoided.2

Implications for your patient if NGS fails

  • Only about 1 in 5 patients are treated based on NGS findings; most treatment decisions are based instead on immunohistochemistry and in situ hybridisation techniques.
  • Caris can also analyse a selection of genes using other techniques if NGS fails.

What can I do if the first specimen fails? 

  • Sometimes we recommend sending a second specimen from the patient. If a new biopsy is needed, state-of-the-art medical devices, such as the Spirotome,3 can help to improve sample success rates.

 

References

  1. Al-Kateb H, Nguyen TT, Steger-May K, et al. Identification of major factors associated with failed clinical molecular oncology testing performed by next generation sequencing (NGS). Mol Oncol. 2015;9(9):1737-43.[URL]
  2. Maetens M, Brown D, Irrthum A, et al. The AURORA pilot study for molecular screening of patients with advanced breast cancer – a study of the breast international group. NPJ Breast Cancer. 2017;3:23. [URL]
  3. Spirotome macrobiopsy for precision medicine [online]. Bioconcise; 2017 [cited May 2018]. Available from: [URL]
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