Solid Tumor Panel with Next Generation Sequencing

The presence of mutations (single nucleotide variants and insertions/deletions) in a panel of 26 cancer genes is detected using the Tumor Hotspot MASTRTM Plus kit (Multiplicom) on the Illumina MiSeq Dx instrument. The Tumor hotspot MASTR Plus assay has been optimized for analysis of formalin-fixed, paraffin-embedded (FFPE) tissue (amplicon lengths 121 to 254 basepairs) and allows complete next generation sequencing analysis and reporting in the acceptable time frame of less than 10 business days.

The test allows sequencing of mutational hot spots within the following 26 cancer genes: AKT1, ALK, BRAF, CDNK2A, CTNNB1, DDR2, EGFR (ERBB1), ERBB2 (HER2), ERBB4, FGFR2, FGFR3, H3F3A, HIST1H3B, HRAS, IDH1, IDH2, KIT, KRAS, MEK1 (MAP2K1), MET, NRAS, PDGFRA, PI3KR1, PIK3CA, PTEN, STK11.

The test is performed based on 5 major processes:

  • Manual DNA extraction from 4 to 8 five µm thick tissue sections (4 for resection specimens; 8 for non-resection specimens) obtained from formalin-fixed, paraffin-embedded solid tumor tissue containing at least 10% tumor cells (Cobas DNA Sample Preparation Kit).
  • Gene target regions are amplified in 4 separate multiplex PCR reactions per individual.
  • Using a universal PCR, amplicons are tagged with specific molecular identifiers and adaptors required for sequencing.
  • Purified and individually tagged amplicon libraries are pooled and further processed by bridge amplification and sequencing using the sequencing by synthesis technology (Illumina MiSeq).
  • Generated sequencing reads are aligned to a reference sequence to identify differences between the obtained sequence reads and the reference. To do end, two bio-informatics pipelines are combined into a superior pipeline (SeqNext, JSI and Sophia DDM, limit of detection 2.5% variant allele frequency). Variants are compared with public and/or private databases in order to identify mutations associated with health and disease. Identified variants are being reported together with their clinical relevance.

 

Clinical implications

The Tumor Hotspot MASTRTM Plus kit allows sequencing of mutational hot spots within 26 cancer genes. The regions of interest are selected ("targeted") for sequencing. By selecting a priori a set of genes with clinical actionability in diverse tumor types, testing utility is maximized and the potential for off-target, incidental findings is minimized. The panel of tumor genes present in the current test kit is in part based on the INCa panel, developed and recommended by the Institut National du Cancer or INCa in France.

Identified variants are being reported together with their clinical relevance. Based on the information obtained from different databases and current literature, observed variants are classified according to the following scheme (Hagemann IS et al. Cancer Genetics 2014;206, 420-431):

1: Predictive/prognostic in patient’s tumor type

2: Predictive/prognostic in another tumor type

3: Reported in Cancer

4: Variant of Unknown Significance (VUS)

5: Most likely benign

For colorectal carcinoma, non-small cell lung cancer and melanoma, those actionable target genes that are considered imperative to be tested for reimbursement of targeted treatments are summarized on page 1 of the final report (KRAS/NRAS, EGFR, BRAF). A complete overview of all findings is given on subsequent pages. Classification of variants other than the actionable target genes mentioned above may be preliminary and re-classification may be required after discussion in a multidisciplinary setting.

Specimen Requirements

Acceptable specimens for this assay are formalin-fixed, paraffin-embedded solid tumor specimens (Colorectal cancer, Melanoma and non-small cell lung cancer). Ideally, fixation time is 6 to 48 hours.

Volume

1 representative paraffin block is preferred. Alternatively, 5 µm thick unstained tissue sections are required (4 sections for resection specimens; 8 sections for non-resection specimens such as small biopsies).

Storage and Shipment Instructions

Maintain and ship specimens at ambient temperature.

Limitations

Insufficient tumor content (<10% tumor cells) and/or poor DNA quality may not allow full coverage of all target regions (for 107/112 exons, 500X mean read depth must be achieved). Tumor content is evaluated prior to analysis and macrodissection is performed to enrich the sample for tumor cells.

The following quality parameters are reported:

  • Tumor cell percentage: must be 10% or higher.
  • DNA quality coefficient: DNA quality is assessed by QC plex (Multiplicom). The assay is based on a standardized multiplex PCR assay that can be used to determine the global FFPE derived DNA quality. The multiplex PCR generates 6 PCR fragments of different lengths (100, 200, 300, 400, 600 and 700 bp) directly assessing the DNA integrity. A quality coefficient > 5 indicates excellent DNA quality, a value of 5.0 – 1.1 good quality, 1.0 – 0.2 acceptable quality and < 0.2 poor quality. Based on the results of DNA quality assessment, 20 ng (excellent DNA quality) – 500 ng (acceptable to poor DNA quality) of DNA input is required.
  • Total Mean Read Depth: the minimal number of total reads per sample must be 185.000 and should ideally be higher than 220.000.
  • Q-score: a base quality score is given for each nucleotide. The average base quality score of a sample must be > 30.

The limit of detection of the test is set at 2.5% variant allele frequency (VAF). For variants with a variant allele frequency between 2.5 -10% (low frequency variants), additional criteria are put in place regarding the required number of variant reads:

  • Sophia DDM variants with at least "VAF * 1250" variant reads when the variant allele frequency is between 2.5% and 5% and with at least "VAF * 422" variant reads when the variant allele frequency is between 5% and 10%.
  • SeqNext variants with at least "VAF * 2500" variant reads when the variant allele frequency is between 2.5% and 5% and with at least "VAF * 844" variant reads when the variant allele frequency is between 5% and 10%.
  • KCE Report 240. Next Generation Sequencing gene panels for Targeted Therapy in oncology and hemato-oncology.
  • Bennett NC and Farah CS. Cancers 2014; 6, 2296-2312. Next Generation Sequencing in Clinical Oncology: Next steps towards Clinical Validation.
  • Aziz N et al. College of American Pathologists’ Laboratory Standards for Next-Generation Sequencing Clinical Tests. College of American Pathologists 2014.
  • Nirav R Shah, Sue Kelly. Next Generation Sequencing (NGS) guidelines for somatic genetic variant detection. New York state department of Health. January 2014.
  • Murray DC, Coghlan ML and Bunce M. PloS One 2015; 10(4). From Benchtop to Desktop: Important Considerations when Designing Amplicon Sequencing Workflows.
  • Hagemann IS et al. Cancer Genetics 2014;206, 420-431. Design of targeted, capture-based, next generation sequencing tests for precision cancer therapy.

 

Special Requirements

None

Turn-Around Time

Ten business days, starting from the first business day after sample receipt at HistoGeneX. 

References

  • KCE Report 240. Next Generation Sequencing gene panels for Targeted Therapy in oncology and hemato-oncology.
  • Bennett NC and Farah CS. Cancers 2014; 6, 2296-2312. Next Generation Sequencing in Clinical Oncology: Next steps towards Clinical Validation.
  • Aziz N et al. College of American Pathologists’ Laboratory Standards for Next-Generation Sequencing Clinical Tests. College of American Pathologists 2014.
  • Nirav R Shah, Sue Kelly. Next Generation Sequencing (NGS) guidelines for somatic genetic variant detection. New York state department of Health. January 2014.
  • Murray DC, Coghlan ML and Bunce M. PloS One 2015; 10(4). From Benchtop to Desktop: Important Considerations when Designing Amplicon Sequencing Workflows.
  • Hagemann IS et al. Cancer Genetics 2014;206, 420-431. Design of targeted, capture-based, next generation sequencing tests for precision cancer therapy.