Next Generation Sequencing (NGS) for Myeloid neoplasms

​Next generation sequencing (NGS) permits the simultaneous investigation of multiple genes.   A targeted NGS panel is now in use fo the detection of single nucleotide variants, small insertions and deletions within clinically relevant genes for patients diagnosed with myeloid malignancies (Table 1).   In addition, the panel is able to detect some clinically relevant gene fusions (Table 2).  NGS is performed using the ThermoFisher Oncomine Myeloid Research Panel on the Thermofisher Ion GeneStudio S5 instrument. Analysis of results is performed using the ThermoFisher Torrent Suite and Ion Reporter software, and reported with respect to an appropropriate reference genome.

Patients who fulfill the following criteria may be eligible for testing:

• Treatment eligible patient with acute myeloid leukaemia
• Transplant eligible patient with a diagnosis of myelodysplastic syndrome
• Transplant eligible patient with a diagnosis of primary myelofibrosis
• For differentiation of hypoplastic MDS and aplastic anaemia
• Atypical myeloproliferative neoplasm (“triple negative” PMF, MDS/MPN overlap)
• Relapse AML patient to provide therapeutic information

Please specify to which category the patient belongs when requesting.

Sample Requirements

Myeloid NGS Testing is performed on both DNA and RNA extracted from bone marrow or blood samples. Where possible, the diagnostic bone marrow specimen should be tested.  Samples collected post-therapy are often unsuitable due to the low neoplastic cell content.

Please complete the Myeloid NGS form: 

Genes/ Fusions assessed

 The following genes and fusion partners are currently available for assessment:

Table 1: Gene regions analysed

Table 2: Fusion transcripts analysed: the most common fusion transcripts involving these partners will be identified.

 Assay Limitations

The ThermoFisher Oncomine Myeloid Research Panel is designed to target clinically relevant regions of the target genes; entire genes are not always analysed.  Therefore, the panel may not detect, or detect with reduced sensitivity, some variants located outwith the target regions. A full list of reference gene transcripts and specific target regions are available on request.

The limit of detection (LoD) of the ThermoFisher Oncomine Myeloid Research Panel has been calculated to be 5% for small nucleotide variants and approximately 10% for insertions and deletions.  Variants present at an allele frequency below the LoD will not expected be detected.  The likelihood of missing a low level variant increases in samples with a low neoplastic cell content.

Due to the wide range of regions/amplicons analysed using this NGS panel, it would not be feasible to calculate a limit of detection for every possible variant.  Differences in the LoD may occur in some genomic regions due to factors intrinsic to DNA structure.  Furthermore, the NGS panel may fail to accurately characterise variants located in long homopolymer regions due to limitations in the sequencing technology.  Sequences that are GC rich and areas that form secondary structures may also prove challenging to sequence.  Failure to detect variants located at the end of amplicons cannot be excluded. 

Samples are reported as having “optimal coverage” where there are a minimum of 250 reads for all reportable amplicons. The Association of Molecular Pathology and the College of American Pathologists recommend a minimum of 250 reads per tested amplicon, corresponding to a false positive or false negative rate of <0.5% when there is a 5% limit of detection (Somak et al., 2017).  Please note, for most samples the read depth is significantly higher than 250 across all target regions.

Reporting of Results 

 Variants are reported according to national and international guidelines. Benign or likely benign variants are not reported, Variants of uncertain significance are ordinarily not reported. It is accepted that variant interpretation may change as new evidence becomes available, hence the interpretation is relevant at the time of reporting. Variants (at both the nucleotide and amino acid level) are reported using HGVS nomenclature.

After reporting, each case will be discussed at a "molecular MDT" which involves a clinical scientist, haematologist and (if required) clinical geneticist. After this meeting, a supplementary report will be issued.

The NGS panel is designed for the detection of somatic variants only.  Whilst germline variants may be present, it is not possible to differentiate between germline and somatic variants using this assay. If a variant is observed in the CEBPA, RUNX1, GATA2 or TP53 (for patients less than 30) genes at an allele frequency that would be consistent with it being germline in origin, this will be highlighted in the report.  In these situations, the case will be discussed with representatives from Molecular and Clinical Genetics.

Turnaround Time

NGS results are expected to be available within 6 weeks of sample receipt.  If testing is urgent please contact the laboratory to discuss.

Accreditation Status

NGS testing using the ThermoFisher Oncomine Myeloid Research Panel and is fully accredited to ISO 15189:2012.

References

Li et al. The Journal of Molecular Diagnostics, 2017 Volume 19 , Issue 1 , 4 – 23.

Roy et al.  The Journal of Molecular Diagnostics, 2017 Volume 20, Issue 1, 4 - 27

Richards et al. Genetics in Medicine, 2015 Volume 17, Issue 5, 405-424