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Frequently asked questions: next-generation sequencing (NGS)

  • What is NGS?

    High-throughput parallel sequencing of multiple segments simultaneously; allows for analysis of multiple target genes or transcripts in a
    single test1,2

    • Conducted using any sample of DNA or high-quality RNA1,4
    • Used for detection of a broad range of targetable and non-targetable mutations and transcripts, including various mutations and fusions1–4
    • Minimum of 24 hours between sample receipt and data; in practical use turnaround times may be substantially longer2
       

    References:

    1. Serrati S, De Summa A, Pilato B, et al. Next-generation sequencing: advances and applications in cancer diagnosis. OncoTargets Ther 2016;9:7355–7365.
    2. Ross J, Cronin M. Whole cancer genome sequencing by next-generation methods. Am J Clin Pathol 2011;136:527–539.
    3. Tsao MS, Hirsch FR, Yatabe Y, eds. IASLC Atlas of ALK and ROS1 testing in lung cancer. International Association for the Study of Lung Cancer. Editorial Rx Press. 2016.
    4. Lipson D, Capelletti M, Yelensky R, et al. Identification of new ALK and RET gene fusions from colorectal and lung cancer biopsies. Nature Med 2012;18:382–384.
  • How does the analytical NGS workflow look like?

    Analytical phase: Next Generation Sequencing


    With permission of Fernando Lopez-Rios, ECP 2017, Pfizer Symposium

  • Briefly, can you tell me what are the benefits and key points to consider when using NGS in clinical molecular diagnostics?


    References

    1. Serrati S, De Summa A, Pilato B, et al. Next-generation sequencing: advances and applications in cancer diagnosis. OncoTargets Ther 2016;9:7355–65.
    2. Luthra R. Chen H. Roy-Chowdhuri S. et. al. Next-generation sequencing in clinical molecular diagnostics of cancer: advantages and challenges. Cancer 2015;7:2023-36
  • What are the differences between DNA and RNA sequencing?

    DNA sequencing includes WGS, WES and targeted sequencing1

    • WGS sequences the whole genome, requiring a large DNA sample, that may be too costly and time consuming for routine practice
    • WES focuses on sequencing the coding regions (exons) of the genome, which represent ~2.5% of the genome, to discover rare or common variants associated with a disorder or phenotype. WES is less costly and faster than WGS
    • Targeted sequencing focuses on genes of interest for a specific disease, and may be an appropriate choice in terms of time and cost for many laboratories

    RNA sequencing requires extracted RNA to be reverse-transcribed into cDNA and amplified1

    • Allows detection of alternative gene-spliced transcripts, post-transcriptional modifications, gene fusions, mutations, single-nucleotidepolymorphisms and changes in gene expression
    • C ommercially available sequencing platforms offer specific kits containing primer sequences for a range of diseases, including lung cancer

    References:

    1. Serrati S, De Summa A, Pilato B, et al. Next-generation sequencing: advances and applications in cancer diagnosis. OncoTargets Ther 2016;9:7355–65.
  • How much will integrating NGS into my lab cost?

    • Estimated costs of incorporating NGS testing for NSCLC for four possible options, including small or medium targeted gene panels on the Illumina Miseq or Hiseq is provided below (Table 1)
    • The estimated cost per sample is dependent on the number of samples per run (Figure 1); a higher number of samples per run results in lower costs per sample, due to the fixed costs per year and fixed costs per run1
    • NGS initiation into the lab is only cost-effective if at least 10 cases are analysed per week

    Challenges with costs, when integrating NGS into routine clinical practice, may be overcome using the following practices:
             a. Consider sharing instruments with other labs in the same hospital
             b. Seek funds through research projects
             c. Collaborate with referral centres; consider involving them in publications as incentive to work together
             d. Work with manufacturers to avoid any hidden costs (e.g. maintenance costs)
             e. Consider long term and rental contracts (this may mean less investment and upkeep is required and upgrading the system may be easier)

    Figure 1. Steps to consider when introducing NGS into your lab.


    The costs are shown for both the small and medium TGP on the Illumina Miseq or Hiseq. There is a maximum of four samples for the medium TGP on the
    Illumina Miseq; therefore, only one data point is given. TGP = targeted gene panel.



    Reference:

    1. van Amerongen RA, Retèl VP, Coupé VM, et al. Next-generation sequencing in NSCLC and melanoma patients: a cost and budget impact analysis. Ecancermedicalscience. 2016;10:684.
  • What are the key considerations for using NGS?

    The implementation of NGS technology can be visualised as four broad steps (Figure 1).1
    Although there are many advantages of using NGS over other traditional sequencing methods, some challenges do exist:

    1. Neoplastic cell, stromal and necrotic content can be highly variable within a sample1

              a. Careful screening and selection of tumour tissue, avoiding necrotic areas, by an experienced pathologist is highly recommended2

    1. NGS workflow consists of several steps

              a. Please see Figure 2 as guidance when implementing NGS into your lab3

    1. High quantity of data is generated, which requires filtering of redundant information to accurately interpret results.3 To try and overcome this issue labs could:

              a. implement regular specialist training (e.g. by NGS company) and/or have dedicated NGS personnel
              b. compare results of previous cases using the new technology
              c. share protocols with other labs using the same platform
              d. conduct research and publish data to help identify any correlation between test results and bioinformatics read outs
              e. enrol in a quality control assessment programme
              f. seek funds through research projects
              g. participate in molecular tumour board (also for referral centre)
              h. participate in intra-lab molecular board before sign out
              i. outsource interpretation to external companies (e.g. Sophia Genetics, Switzerland)

    1. Long-term storage of data, without breaking any data protection laws, is required4

    References:

    1. Deans ZC, Costa JL, Cree I, et al. Integration of next-generation sequencing in clinical diagnostic molecular pathology laboratories for analysis of solid tumours; an expert opinion on behalf of IQN Path ASBL. Virchows Arch 2016;470:5–20.
    2. Tsao MS, Hirsch FR, Yatabe Y, eds. IASLC Atlas of ALK and ROS1 testing in lung cancer. International Association for the Study of Lung Cancer. Editorial Rx Press. 2016.
    3. Singh RR, Luthra R, Routbort MJ, et al. Implementation of next generation sequencing in clinical molecular diagnostic laboratories: advantages, challenges and potential. Expert Rev Precis Med Drug Dev 2016;1:109–20.
    4. Serrati S, De Summa A, Pilato B, et al. Next-generation sequencing: advances and applications in cancer diagnosis. OncoTargets Ther 2016;9:7355–65.
  • What control do I need to use when using NGS?

    • NGS should be validated in the local laboratory environment prior to clinical implementation, and before the use of new gene panels
    • A range of known mutations and commercially available controls can be used for the validation process
    • A range of mutation types and allelic frequencies should be tested to provide assurance that they will be detected if present in clinical samples
    • The NGS test should also be verified to ensure it meets the predefined performance specifications
    • Always recommended to use a positive control sample for clinical runs

    Reference:
    Deans ZC, Costa JL, Cree I, et al. Integration of next-generation sequencing in clinical diagnostic molecular pathology laboratories for analysis of solid tumours; an expert opinion on behalf of IQN Path ASBL.
    Virchows Arch 2016;470:5–20.

  • What NGS technologies are currently available and how do they work?

    Sequencing platforms

    • Two main NGS platforms are currently available (Figure 1)
    • Illumina sequencing1–3
      – The error rate estimated for Illumina technology is <0.4%4
    • Ion Torrent from Thermo Fisher Scientific
      – The error rate is 1.8–1.9%, mostly in the detection of homopolymer stretches4
       

    Figure 1. Most common sequencers and their characteristic



    References

    1. Ross J, Cronin M. Whole cancer genome sequencing by next-generation methods. Am J Clin Pathol 2011;136:527–539.
    2. Liu L, Li Y, Li S, et al. Comparison of Next-Generation Sequencing Systems. J Biomed Biotechnol. 2013;251364.
    3. EMBL-EBI. EBI: next generation sequencing practical course – what is next-generation sequencing. 2012. http://www.ebi.ac.uk/training/online/course/ebi-next-generation-sequencing-practical-course/what-you-will-learn/whatnext-
    4. generation-dna- [Accessed February 2017].
    5. Serrati S, De Summa A, Pilato B, et al. Next-generation sequencing: advances and applications in cancer diagnosis. OncoTargets Ther 2016;9:7355–7365.
    6. Kamps R, Brandão RD, van den Bosch BJ, et al. Next-Generation Sequencing in Oncology: Genetic Diagnosis, Risk Prediction and Cancer Classification. Int J Mol Sci. 2017;18:308.

     

  • What are the key considerations when handling samples for NGS?

    key limiting factor in NGS is the quality and representative nature of the samples.1 Thus, a number of specific sample handling processes are advised:

    1. Minimise sample loss
    • To minimise the loss of material, the following are suggested:2,3
      – Sufficient multidisciplinary communication, between oncologists and pathologists, so the paraffin block is cut as few times as possible2
      – Reasonable use of classificatory immunohistochemistry with a restricted panel ofantibodies2
      – Separately embed tissue fragments into individual blocks to examine singularly and reduce tissue wastage; ensure they are shallow facing (Figure 1)
    1. Sample transportation, handling and storage1,4
      – Use neutral buffered formalin
      – Calibrate the length of time of tissue fixation to the size of specimen
      – Minimise the risk of cross contamination during the cutting of sections by replacing the knife regularly, avoiding water baths and using disposable plastic ware
      – Avoid decontamination procedures that compromise nucleic acid
      – In cases where tissue material is lacking, cytological material that has been successfully tested for genotype analysis is recommended for molecular analyses to provide a high yield of quality DNA
    2. Sample morphology and region of interest
    • To ensure the correct interpretation of NGS results, select an area of the sample for extraction that contains the highest proportion of neoplastic cells and that exceeds the minimum level of tumour content for NGS detection, whilst avoiding necrotic tissue; document the neoplastic fraction4
    1. Sample misidentification
    • Procedures to minimise the risk of patient misidentification must be employed:1
      – Barcoding may be used, and should be introduced at the earliest opportunity in the pathway
    • An end-to-end risk analysis of the misidentification of samples should be performed before introducing NGS, and regularly during operation1

    Figure 1. Schematic representation of the approach used for ‘molecular priority’ specimen processing inwhich tissue fragments are separately embedded and shallow faced3

    Shallow facing may result in inability to visualise the complete tissue cross-section (represented by dotted line); however superficial facing is often sufficient to evaluate the presence of tumour. Abbreviations: FISH, fluorescence in situ hybridization; IHC, immunohistochemistry


    References

    1. Deans ZC, Costa JL, Cree I, et al. Integration of next-generation sequencing in clinical diagnostic molecular pathology laboratories for analysis of solid tumours; an expert opinion on behalf of IQN Path ASBL. Virchows Arch 2016;470:5–20.
    2. Conde E, Angulo B, Izquierdo E, et al. Lung adenocarcinoma in the era of targeted therapies: histological classification, sample prioritization, and predictive biomarkers. Clin Transl Oncol 2013;15:503–508.
    3. Aisner D, Rumery M, Merrick D, et al. Do More With Less. Arch Pathol Lab Med. 2016;140:1206–20.
    4. Tsao MS, Hirsch FR, Yatabe Y, eds. IASLC Atlas of ALK and ROS1 testing in lung cancer. International Association for the Study of Lung Cancer. Editorial Rx Press. 2016.

     

  • What is involved in the data analysis of NGS?

    Bioinformatics tools used for processing, aligning and detecting variants in NGS data are commonly referred to as the data analysis or bioinformatics pipeline (Figure 1). Stages in the bioinformatics pipeline include:1,2

    • Read processing/quality: performed by the sequencer
    • Alignment: alignment of sequencing reads to the genome of reference (e.g. the human genome)
    • Variant calling: key challenge for variant caller algorithms is to distinguish sequencing errors from real variation; therefore, the more times a variant is sequenced, the more reliable the call
    • INsertion/DELetion (indels): second most common type of genomic variation, and of clinical relevance in genes such as EGFR. Reliable identification of indels by software packages has proven challenging
    • Variant annotation: determines if a sequence variant is false or true and if the functional interpretation of that variant is related to the gene (protein) function
    • Variant interpretation: biological interpretation of the true variants

    A wide range of software is available to analyse NGS data, and the major sequencing platform manufacturers also supply software to complement their machines. It is also possible to outsource interpretation of results to certain companies (e.g. Sophia Genetics, Switzerland). It is advised to have regular molecular tumour board meetings in order to explain and discuss patient results and also provide recommendations for individual patient treatments.

    Figure 1. Example of DNA-seq bioinformatics pipeline for Illumina2


    References

    1. Deans ZC, Costa JL, Cree I, et al. Integration of next-generation sequencing in clinical diagnostic molecular pathology laboratories for analysis of solid tumours; an expert opinion on behalf of IQN Path ASBL. Virchows Arch2016;470:5–20.
    2. Kamps R, Brandão RD, van den Bosch BJ, et al. Next-Generation Sequencing in Oncology: Genetic Diagnosis, Risk Prediction and Cancer Classification. Int J Mol Sci. 2017;18:308.
  • When is quality assessment required?

    The College of American Pathologists (CAP) has published the molecular pathology checklist, which includes quality control recommendations for the whole NGS process1

    • External quality assessment (EQA) schemes are imperative and should be used wherever possible; inter-laboratory comparisons may offer a suitable alternative2
    • Laboratories should have written policies for the selection and evaluation of reference laboratories1
    • The Phred quality score system is a widely accepted method of assessing the quality of sequencing data including NGS, and can be used to compare sequencing methods and platforms2

    References

    1. Aziz N, Zhao Q, Bry L, et al. College of American Pathologists’ Laboratory Standards for Next-Generation Sequencing Clinical Tests. Arch Pathol Lab Med 2015;139:481–493.
    2. Deans ZC, Costa JL, Cree I, et al. Integration of next-generation sequencing in clinical diagnostic molecular pathology laboratories for analysis of solid tumours; an expert opinion on behalf of IQN Path ASBL. Virchows Arch 2016;470:5–20.
  • Reporting of NGS results

    • Reporting of NGS results should follow the general principles of clinical reporting, providing pertinent information clearly and concisely, and should sit in line with international diagnostic standards (e.g. ISO 15189) and professional guidelines1
    • The NGS patient report should be one, or no more than two, pages in length1
    • Laboratories should have a policy for reporting incidental genetic findings unrelated to the clinical purpose of testing
    • Ethical considerations must also be taken into account when deciding whether to reveal certain genetic information2
    • Over-reporting should be avoided- notify the clinician of requested results with further information available upon request

    References

    1. Deans ZC, Costa JL, Cree I, et al. Integration of next-generation sequencing in clinical diagnostic molecular pathology laboratories for analysis of solid tumours; an expert opinion on behalf of IQN Path ASBL. Virchows Arch 2016;470:5–20.
    2. Aziz N, Zhao Q, Bry L, et al. College of American Pathologists’ laboratory standards for next-generation sequencing clinical tests. Arch Pathol Lab Med 2015;139:481–93.
  • What should I include in a patient report when recording NGS results?

    • Patient identifiers
    • Sample type (e.g. FFPE, fresh frozen)
    • Tissue/tumour type (e.g. lung, colorectal, melanoma)
    • Tissue sample identification (e.g. unique block number)
    • Restatement of the clinical question
    • Percentage of neoplastic content of sample used for NGS
    • Extent of testing performed (i.e. which genes and which regions)
    • NGS method used (platform, type of panel, etc.)
    • Sensitivity of the method
    • Reference sequences for genes tested
    • Results (using HGVS mutation nomenclature)
    • How/where additional information about the analysis can be obtained
    • Interpretation and conclusion

    Reference

    1. Deans ZC, Costa JL, Cree I, et al. Integration of next-generation sequencing in clinical diagnostic molecular pathology laboratories for analysis of solid tumours; an expert opinion on behalf of IQN Path ASBL. Virchows Arch 2016;470:5–20.
  • Should I classify the NGS results we obtain?

    NGS results can help in the diagnosis, predict prognosis or guide optimal therapy of a patient; it can therefore be helpful if results are classified and reported according to their potential for clinical management. The clinical utility of a given result can fall into the following three categories:

    1. Clinical: abnormalities that have a current approved/licensed therapeutic indication or are used for diagnosis, prognosis or therapeutic monitoring
    2. Clinical trials: abnormalities that are hypothesised to predict response to a novel compound; entry into a clinical trial may be possible
    3. Research: mutations not currently used to inform clinical management, but which have a biological effect in tumour oncogenesis which may have a future clinical utility

    When assigning abnormalities to the above categories, use context of the tumour/tissue type as resulting clinical action will vary e.g. abnormalities classified as category 2 in a particular tumour type could fall into category 1 in another tumour type.


    Reference
    Deans ZC, Costa JL, Cree I, et al. Integration of next-generation sequencing in clinical diagnostic molecular pathology laboratories for analysis of solid tumours; an expert opinion on behalf of IQN Path ASBL. Virchows Arch
    2016;470:5–20.

  • Which biomarkers have clinical utility in NSCLC?

    What Biomarkers Have Clinical Utility in NSCLC

     

    Although a broad range of biomarkers have been examined inNSCLC, there are several that have particular clinical utility in this disease state.

    Biomarker

    Molecular Diagnostic Target

    ALK*

    Chromosomal translocation and fusion of ALK gene

    ROS*

    Chromosomal translocation and fusion of ROS1 gene

    EGFR*

    Activating mutation within intracellular catalytic domain of EGFR

    BRAF *

    Activating mutation, especially in position V600E

    PD-1/PD-L1

    Overexpression of PD-1/PD-L1

    MET**

    1.High MET amplification MET:CEP7 ratio >5 resulting  in overexpression and kinase activity activation

    2.Splice-site alterations and point mutations resulting in RNA splicing-based skipping of MET exon 14 and in activation of MET kinase activity

    KRAS**

    Activating mutation within catalytic RAS domain

     

    *Drugs are currently available that are indicated for these patients

    ** Investigational; no drugs with formal indication for these patients

     

     

     

    Adopted from Korpanty GJ et al., Biomarkers That Currently Affect Clinical Practice in Lung Cancer: EGFR, ALK, MET, ROS-1, and KRAS.Front Oncol. 2014 Aug 11;4:204.

  • What are currently available NGS panels and how do they differ?

    Comparison of NGS panels and library preparation kits

     

     

    Minimum DNA Input

    Sample Types

    Content and target

    Target or Amplicon Size

    Library Preparation Time

    Cost

    Illumina TruSeq® Amplicon Cancer Panel

    150 ng
    250 ng

    High quality gDNA, FFPE

    212 amplicons for 48 genes

    170-190 bp

    Fewer than 7 hours

    11,769 euros ($13,428) for 96 samples

    Illumina TruSeq® Custom Amplicon v1.5

    50 ng

    Fresh, frozen, or FFPE

    Up to 1,536 amplicons (custom number of hotspots)

    150, 175, 250, and 425 bp

    10 hours

    Dependent on number of amplicons

    Illumina TruSeq® Custom Amplicon Low Input Library Prep Kit

    10-50 ng (depending on FFPE DNA quality)

    Low input samples FFPE

    Up to 1,536 amplicons (custom number of hotspots)

    150, 175, and 250 bp

    6.5 hours

    Dependent on number of amplicons

    Illumina TruSight® Cancer Sequencing Panel

    50 ng

    gDNA (FFPE compatibility not supported)

    ~4,000 probes for 1,700 exons on 94 genes and 284 SNPs

    Cumulative target region: 255 kb.Individual region size enriched: 350-650 bp

    1.5 days

    Dependent on number of targets

    Illumina TruSight® Tumor 15

    20 ng

    FFPE

    250 amplicons for 15 genes

    ~150-175 bp on average

    7 hours

    2,354 euros ($2,686) for 24 samples

    Illumina TruSight® Tumor 26

    30-300 ng (depending on FFPE DNA quality)

    FFPE

    174 amplicons for 26 genes

    165-195 bp

    Fewer than 7 hours

    5,885 euros ($6,715) for 48 samples

    Ion AmpliSeq™ Cancer Hotspot Panel v2 (with primer pool)

    10 ng

    FFPE and Fine Needle Aspirates (FNA) (among others)

    207 amplicons for 50 genes

    111-187 bp

    3.5 hours

    Ion AmpliSeq™ Cancer Hotspot Panel v2: 216 euros ($246) for 8 reactions
    Ion AmpliSeq™ Library kit 2.0: 860 euros ($980) for 8 reactions

    Ion AmpliSeq™ Comprehensive Cancer Panel

    40 ng

    FFPE and Fine Needle Aspirates (FNA) (among others)

    16,000 amplicons for more than 400 genes

    125-175 bp

    3.5 hours

    860 euros ($980) for 8 reactions

    Roche GS FLX Titanium Rapid Library Preparation Kit

    500 ng

    Double stranded DNA

    N/A

    3 kb, 8 kb, or 20 kb inserts

    Not available

    $1,192 for 12 library preparations

     

     

    Reference: Bennett CW et al., Oncotarget. 2016 Oct 25;7(43):71013-71035