Good practices for NGS panel customization

Cancer remains a leading cause of death worldwide, accounting for almost 10 million deaths in 2020 alone.1 Conventional chemotherapy and radiotherapy treatments have notable limitations and an array of negative side effects.

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Precision medicine is key to developing more effective and less toxic treatments, representing the present and future of oncology research. This approach offers significant promise for incremental improvements in patient outcomes throughout the field of oncology.

Molecular profiling is one of the most fundamental techniques in precision medicine, used for identifying biomarkers and detecting actionable mutations. An improved understanding of the molecular mechanisms underpinning cancerous tumors is crucial for developing precision medicines that target these specific mechanisms, enabling more effective treatments.

Over the past decade, the treatment of advanced non-small cell lung cancer (NSCLC) has increasingly relied on tissue specimens and biomarkers to guide targeted treatment options. The early detection of these biomarkers has allowed medical professionals to avoid treatments that are unlikely to benefit patients.

Numerous biomarker-defined patient subgroups now exist, with evidence suggesting that treatment with targeted and immuno-oncology therapies offers improved clinical outcomes compared to cytotoxic agents.

The National Comprehensive Cancer Network® (NCCN) currently recommends the use of molecular testing in NSCLC practice guidelines, including testing for ALK rearrangements, EGFR mutations, KRAS, ROS1, BRAF, NTRK1/2/3, METex14 skipping, RET, and ERBB2 (HER2). The NCCN also recommends PD-L1 testing via immunohistochemistry (IHC). 2

Several of these biomarker-driven subgroups incorporate a diverse array of approved targeted therapies, with the exception of high-level MET amplifications, which is currently regarded as an emerging NSCLC biomarker.2

Good practices for NGS panel customization

Image Credit: Cerba Research

Next-generation sequencing in oncology

Current precision oncology primarily relies on the capacity of next-generation sequencing (NGS) to simultaneously analyze multiple genetic aberrations, for example, small insertions/deletions (indels), single nucleotide variants (SNVs), and copy number variants (CNVs).

Information on tumor mutational status was limited before the development of NGS because traditional Sanger sequencing and PCR methodologies could only analyze a single gene at a time. These methods are useful for performing assays to isolate a specific, known target gene, but discovering new target genes through these methods is incredibly time-consuming.

There may also be a need to use rebiopsies to investigate other single genes, making Sanger sequencing and PCR methodologies relatively impractical for the identification of new gene alterations.

NGS can, however, analyze millions of genetic segments in parallel, facilitating the sequencing of multiple cancer-driving genes in a single assay while offering increased sensitivity and requiring only limited availability of cancerous tissue.

Table 1 summarizes the benefits and shortcomings of these methods and highlights how, when used together, these methods can complement one another to ensure an optimized clinical outcome.

Table 1. Summary of the benefits, uses, and disadvantages of NGS and Sanger sequencing in oncology precision medicine. Source: Cerba Research

  Advantages Disadvantages
NGS Account for locus heterogeneity
Accurately and sensitively analyze multiple genes in one assay with limited tissue.
Fast and less labor-intensive method
Increased chance of missing relevant mutations
Prone to sequencing artifacts
Increased false positive rate
Sanger sequencing Offers precise confirmation of genetic variants of clinical significance
Low false positive rate
Useful to fill in regions that have failed to amplify in complex sequences
Does not account for locus heterogeneity
Can only analyze one gene in an assay, therefore tissue continues to be the issue.
Time-consuming and labor-intensive

 

Since realizing its clinical potential, NGS has been employed in a wide range of oncology clinical trials and is currently seeing relatively successful implementation in clinical practice. Its capacity for broad molecular profiling is a key factor in improving patient care.2

NGS allows the genome of individual cancer patients to be sequenced, enabling the identification of genetic changes related to cancer. However, processing the vast amount of data from whole genome sequencing is extremely challenging, impractical, and too costly for routine patient diagnosis.

These challenges are being addressed with recent developments leading to high-throughput assays of customized NGS-based panels. These panels can examine clinically relevant genes, allowing rapid, cost-effective investigation of genomic abnormalities linked to specific cancers and disease types.

NGS-customized broad molecular profiling can sequence at a deeper level, enabling more precise detections at a reduced cost. Broad-based genomic testing is crucial for various clinical applications, especially the provision of personalized and precisely targeted therapies through the accurate and efficient identification of potentially actionable mutations.

The incorporation of clinically relevant target sequences into these customized panels is central to providing individualized oncology treatment.

This article explores the importance of panel customization assays, assessing the therapeutic value of related treatments and patients’ capacity to respond to potential therapies. The examples provided focus on NGS of formalin-fixed paraffin-embedded (FFPE) samples, as well as more recent work with circulating tumor DNA (ctDNA).

Next-generation sequencing broad panel assays

A range of current precision oncology clinical trials are reliant on customized, targeted NGS-panel assays in order to identify actionable targets. Customized NGS panels ranging from 20 to more than 500 genes enable users to reliably and rapidly identify the genetic aberrations most commonly associated with a specific cancer type.5

NGS-panel assays can achieve a higher depth of coverage using this customized panel design, enabling a lower threshold value for detecting intratumoral heterogeneity and low frequency of variant allele changes. These markers are inherent to the majority of cancers, meaning that their efficient detection is central to precision oncology treatments.6

Cerba Research continues to adapt its oncopanels to meet international guidelines related to solid and liquid tumors. The company is committed to providing the relevant, up-to-date panels necessary for efficient assays and the rapid administration of effective treatments.

Cerba Research’s genomic experts remain on hand to support its customers and will accurately customize panels or make these more visible to assays where required.

By leveraging the company’s customized panels in NGS assays, researchers can focus on the genes most pertinent to their research, improving the detection of actionable variants and the identification of potential drug targets.

Cerba Research provides its customers with the tools to examine both the number of somatic mutations in a given DNA sequence and the ability to ascertain the number of alterations present in one or more nucleotides.

The company’s expertise, state-of-the-art capabilities and extensive history in genomics have enabled the development of robust multiplex panel assays, as well as broad-panel assays designed to optimize analysis of patient FFPE samples.

NGS panels from Cerba Research are able to detect a wide range of cancerous hallmarks, including indels, SNVs, and CNVs. The company’s broad panel NGS assays can also be used to highlight the tumor mutational burden (TMB), homologous recombination deficiency status (HDR), and microsatellite instability (MSI) status of patient samples.

Customized panels deliver more efficient, sensitive data collection, simplifying, streamlining and speeding up the bioinformatic processes necessary to analyze collected data.

A large variety of oncopanels are available at Cerba Research’s international laboratories, and support is available for researchers who need help selecting the most suitable panel for their specific oncology clinical trial. Assuming the available NGS panels meet a customer’s trial requirements, Cerba Research can often provide a rapid turnaround time of 10-15 days.

An entirely new NGS panel can be created upon request, or extra genes can be added to an existing panel. It is important to note, however, that this degree of customization typically requires a three-month fit-for-purpose validation turnaround time.

This level of efficiency means that patients can receive faster-targeted treatment, an essential factor when studying an advanced disease with low survival rates.

Not only are customized panel assays important for NGS-based oncology molecular profiling, but these are also key to enhancing the efficiency of immunohistochemistry (IHC) assays. These assays can be used to assist NGS assays in a reflex fashion, for example, reflex PD-L1 stains.

IHC assays can also be used to verify co-expression and spatial organization in multiple targets. This method complements NGS assays, characterizing tumors to identify biomarkers that could predict a patient’s response to immunotherapy.

Both multiplex and simplex IHC can help facilitate the progression from preclinical to clinical trials because this approach better validates therapeutic targets while characterizing treatment efficacy and helping inform patient selection for a specific treatment or clinical trial option.

Cerba Research’s diverse array of customized NGS and IHC panels can support the provision of more tolerable and effective treatment options.

Good practices for NGS panel customization

Image Credit: Cerba Research

Table 2 provides detailed insight into the company’s offering in the NSCLC biomarker space, highlighting the solutions’ alignment with different international guidelines. For example, the NCCN guidelines state that “broad-based genomic testing approaches that efficiently utilize limited biopsy tissue while maximizing diagnostic genomic information are most commonly NGS-based”.2

Table 2. What are NSCLC guidelines proposing? Aligned with Cerba Research NGS, IHC, and FISH capabilities. Source: Cerba Research

Lung Cancer Biomarker Most commonly deployed1-4  Additional Assay(s)1 Cerba Research NGS+ Cerba Research IHC+* Cerba Research FISH+
EGFR NGS, RT-PCR Sanger sequencing, single gene X X  
ALK NGS, IHC, Liquid Biopsy FISH (reflex), RT-PCR X X X
ROS1 NGS FISH (reflex), IHC, RT-PCR X X X
BRAF NGS, RT-PCR, Sanger sequencing IHC X X  
KRAS NGS, RT-PCR, Sanger sequencing   X MEK1  
MET NGS, RNA-based NGS   X X X
RET NGS, RNA-based NGS FISH, RT-PCR X X X
NTRK1/2/3 NGS, RNA-based NGS FISH, IHC, PCR X X X
EGFR T790M NGS, Liquid Biopsy   X    
PD-L1 IHC     X  
HER2 NGS Sanger sequencing, targeted PCR X X X

1. NCCN guidelines 2023;
2. Bebb et al. Curr Oncol 2021;
3. Cabillic et al. ESMO Open 2018;3(6):e419;
4. Li et al. J Nat Cancer Center 2021;
Cerba Research Data In-house mostly available through the ACTOnco®/Cerba Paris (NGS) or Cerba Montpellier/NY (IHC) or Cerba Paris (FISH);
*Validation level may vary; IHC=Immunohistochemistry; NGS=Next-generation sequencing; FISH=Fluorescent in situ hybridization.

Selection of NGS broad panel assays

The selected genes are of paramount importance when looking to take full advantage of the capabilities of NGS panels to screen several genetic markers simultaneously.

For example, when performing routine diagnostic assays of samples, it is advisable to use predesigned panels of genes with an established predictive and/or prognostic significance to ensure flexibility when examining tumors from different genetic origins.7

Customized NGS panels are also required to extract information on cancerous tumors’ molecular profiles in order to accurately assess the therapeutic value of a compound and a patient’s potential response to treatment.

Cerba Research’s vast bioinformatic database allows the company to offer a wide range of existing panels. These panels are available worldwide and have been validated in diagnostic laboratories prior to implementation in clinical and routine settings.

NSCLC precision treatment trials represent a prominent example of the benefits afforded by customized panels. Guidelines recommend that a minimum of 20 genes be examined when conducting molecular screening for therapeutic trials in NSCLC treatment; for example, EGFR, BRAF, HER2, KRAS, PI3KCA, NTKR, ALK, MET, AKT1, BRCA1/ BRCA2, HRAS, NRAS, ROSI, RET, MET, FGFR1/2/3, and NOTCH1/NOTCH2.2,8

NCCN also recognizes “that many currently available NGS-based assays used to fully genotype NSCLC are larger than the 50-gene limit threshold.”2

Cerba Research’s customized OncoSign panels are ideally suited to working with these valuable NSCLC genes, and the company’s team of experts can offer advice on the most suitable panel customization for efficient and precise NSCLC tissue assays.

For example, the extra-large Cerba OncoSign 600+ is capable of detecting 638 DNA-based genes, including 20 fusion genes with established, emerging, and exploratory values. This allows for the detection of known mutations in ovarian, breast, colon, melanoma, lung, bladder, GIST, and rare tumors.

It is also possible to determine the MSI, TMB, and HRD status of tumors such as breast (TMB, MSI), ovarian (HRD, MSI, TMB), and prostate cancer (TMB).2

The company’s customized broad-based panels represent a range of efficient, effective, and affordable NGS assays boasting state-of-the-art precision oncology technologies.

Available oncopanels include:

Cerba OncoSign 600+

  • 658 genes
  • 20 RNA (fusions)
  • TMB, MSI, and HRD
  • Analysis of cancer hallmarks on FFPE tissue

Cerba OncoSign

  • 59 genes
  • 42 DNA
  • 17 RNA (fusions)
  • 15 microsatellites
  • HRD status (CE-IVD marked can be performed in a separate panel)
  • Analysis of cancer hallmarks in FFPE tissue

Cerba OncoSign ctDNA

  • 42 DNA
  • 15 microsatellites
  • Analysis of cancer hallmarks on ctDNA (liquid biopsies)

Good practices for NGS panel customization

Image Credit: Cerba Research

Panels can also be customized explicitly to meet specific oncology research requirements or relevant genes can be added to an existing broad panel assay.

The most practical means of producing these panels and developing these novel markers is to leverage conventional assays (for example, Sanger sequencing or pyrosequencing) to screen for single genes.7

Generated novel markers can then be added to the panel and revalidated for implementation. This approach ensures there is minimal need for panel redesign and revalidation.

It has been noted that primer or probe multiplexing increases as new genetic markers are added to panels. This is because target capture techniques tend to be either multiplexed primer- or probe-based, and this is believed to limit the overall performance of conventional panel assays.7

Cerba Research assays are able to hinder the formation of primer dimers when generating the primer design by masking regions in which primers cannot bind.

Customized panels can be generated to accommodate any gene(s) of interest with a real-world validation turnaround time of around three months, streamlining and expediting development and implementation in clinical trial operations.

Circulating tumor DNA (ctDNA)

There are generally only limited supplies of biopsy tissue available from solid tumors, and the acquisition of these requires an invasive procedure. These issues are compounded by around one-fifth of patients not having enough tumor tissue to perform NGS at all.9

Circulating cell-free tumor DNA (ctDNA), or ‘liquid biopsies,’ represents a far less invasive, less painful, lower risk, and easier means of NGS sampling. ctDNAs can be easily extracted by taking patient blood samples because these are released into the bloodstream during the apoptosis or necrosis of tumor cells.10

Cerba Research frequently leverages its expertise in liquid biopsy assays and its range of existing ctDNA-based panels to customize an entirely new panel or add a new gene to an existing panel in line with trial requirements.

A number of ctDNA panels are available. These are listed alongside their instruments and gene numbers (where applicable).

  • Cerba OncoSign ctDNA (Illumina, 59 genes)
  • Cobas® EGFR mutation test (Roche Cobas)
  • EGFR exons 18-21 (Illumina)
  • Rapid EGFR (42 variants/PCR)
  • EGFR, KRAS, BRAF, HER2, MET, STK11 & KEP1 panel (Illumina)
  • TSO500 ctDNA (Illumina)
  • ACTMonitor® + (Ion Torrent, 50 genes)
  • ACTMonitor® Lung (Ion Torrent, 11 genes)
  • ACTMonitor® Colon (Ion Torrent, 13 genes)
  • ACTMonitor® Breast (Ion Torrent, 8 genes)

Conclusion

Cerba Research boasts an extensive portfolio of broad-panel assays developed using both FFPE samples and ctDNA. The company also offers the option to customize panels according to trial needs.

State-of-the-art, efficient, cost-effective assays are available, along with medical-grade reports suitable for clinical trials. These tools are central to the progression of precision oncology trials towards more tolerable and effective treatment options.

Data generated using the company’s customized panels is more beneficial to clinical trials than data acquired via ‘off-the-shelf’ panels, as this allows users to focus on the genes most relevant to their research. This focus results in the acquisition of more clinically valuable data and greater improvements in patient care.

Cerba Research leverages its NGS capabilities, genomics expertise, diverse array of oncopanels, and access to global bioinformatic databases to help improve patient outcomes with precision oncology. Cerba’s team is also always on hand to provide customization options and support clinical trials as required.

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References and further reading

  1. Ferlay, J., Ervik, M., Lam, F., Colombet, M., Mery, L., Piñeros, M., Znaor, A., So- erjomataram, I. and Bray, F. 2020. Global Cancer Observatory: Cancer Today. Lyon: International Agency for Research on Cancer. IARC; 2018.
  2. National Comprehensive Cancer Network Guidelines. Available at https://www. nccn.org/guidelines/category_1. Accessed June 21, 2023.
  3. Pereira, M., Malta, F., Freire, M. and Couto, P. 2017. Application of next-gener- ation sequencing in the era of precision medicine. Applications of RNA-seq and omics strategies: From microorganisms to human health. London, England, UK: In- TechOpen, pp.293-318.
  4. Aggarwal, C., Rolfo, C.D., Oxnard, G.R., Gray, J.E., Sholl, L.M. and Gandara, D.R. 2021. Strategies for the successful implementation of plasma-based NSCLC geno- typing in clinical practice. Nature Reviews Clinical Oncology. 18(1), pp.56-62.
  5. Sims, D., Sudbery, I., Ilott, N.E., Heger, A. and Ponting, C.P. 2014. Sequencing depth and coverage: key considerations in genomic analyses. Nature Reviews Genetics. 15(2), pp.121-132.
  6. Horak, P., Fröhling, S. and Glimm, H. 2016. Integrating next-generation sequencing into clinical oncology: strategies, promises and pitfalls. ESMO open. 1(5), p.e000094.
  7. Singh, R.R., Luthra, R., Routbort, M.J., Patel, K.P. and Medeiros, L.J. 2016. Imple- mentation of next generation sequencing in clinical molecular diagnostic laborato- ries: advantages, challenges and potential. Expert Review of Precision Medicine and Drug Development. 1(1), pp.109-120.
  8. Morganti, S., Tarantino, P., Ferraro, E., D’Amico, P., Viale, G., Trapani, D., Duso, B.A. and Curigliano, G. 2019. Complexity of genome sequencing and reporting: next gen- eration sequencing (NGS) technologies and implementation of precision medicine in real life. Critical Reviews in Oncology/Hematology. 133, pp.171-182.
  9. Next generation sequencing with liquid biopsies (ctDNA). Cerba Research. Avail- able at: https://cerbaresearch.com/solutions/specialty-lab-biomarker-solutions/genomics/ngs-for-liquid-biopsies/. Accessed June 21, 2023.
  10. Diaz, J.L. and Bardelli, A., 2014. Liquid biopsies: Genotyping circulating tumor DNA. Journal of Clinical Oncology. 32(6), pp.579-586.

Acknowledgments

Produced from materials originally authored by Raouf Djebali, M.Sc., and Rania Gaspo, B.Pharm., Ph.D., from Cerba Research.

About Cerba Research

For over 35 years, Cerba Research has been setting the industry standard for exemplary clinical trial conduct. Today, across five continents, with a focus on precision medicine, we are changing the paradigm of the central lab’s role in complex clinical research.

From protocol inception through development and to market, our passionate experts deliver the highest quality specialized and personalized laboratory and diagnostic solutions. Partner with us for the most efficient strategy to actualize your biotech and pharmaceutical products sooner and improve the lives of patients worldwide.


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Last updated: Jan 17, 2025 at 11:25 AM

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