Contributions
Abstract: PB2278
Type: Publication Only
Background
In the West Midlands region of the UK, all patients with a suspected myeloproliferative neoplasm (MPN) have access to quantitative analysis of JAK2 V617F by droplet digital PCR as standard of care. The British Committee for Standards in Haematology recommends that suspected MPN cases have investigation of JAK2 exon 12, CALR and MPL genes if JAK2 V617F is negative.
Aims
The aim of the project was to improve the MPN service by substituting sequential analysis of individual target regions within the JAK2, CALR and MPL genes with a single assay, and to increase the number of genes available for analysis.
Methods
A commercial next generation sequencing (NGS) gene panel (Oxford Gene Technology, SureSeq Myeloid Panel), coupled with the Illumina MiSeq platform was validated and implemented. The gene panel utilises hybridization based enrichment technology and consists of 25 MPN–related genes. During the validation stage the following were enriched and analysed: 29 positive control samples with 30 known pathogenic variants, 30 negative control samples without known pathogenic variants in the JAK2, CALR and MPL genes, and 24 MPN samples of unknown mutational status. Thus so far over 1500 clinical samples have been analysed and reported since the service was introduced in October 2016.
Results
The panel has successfully identified: a large range of known pathogenic variants at high sensitivity (JAK2 V617F variant allele frequency 1%, CALR Type I frameshift variant allele frequency 3%), a potential alternative driver mutation in a known low level JAK2 V617F positive patient, a rare MPL exon 4 pathogenic variant and also the detection of low level CALR pathogenic variants, which would not have been detected by Sanger sequencing analysis. In one patient the panel identified the presence of two different JAK2 exon 14 pathogenic variants in cis (JAK2 V617F and JAK2 C618R). The JAK2 C618R prevented the hybridization of the probe binding site of the JAK2 V617F ddPCR assay which had led to a false negative result by ddPCR. The validation procedure also explored coverage and limits of sensitivity, potential chemistry specific artefacts and identified common polymorphisms for all 25 genes.
Conclusion
The panel has replaced the current sequential analysis of CALR, MPL and JAK2 exon 12 in JAK2 V617F negative patients and reduced turn-around-times with increased accuracy and sensitivity compared to Sanger sequencing and fragment analysis. Our current clinical service operates on a two tier system whereby clinicians can request analysis of the full 25 gene panel or a 4 gene subset (JAK2, CALR, MPL, CBL as an in silico analysis).
Session topic: 16. Myeloproliferative neoplasms - Clinical
Keyword(s): mutation analysis, Myeloproliferative disorder
Abstract: PB2278
Type: Publication Only
Background
In the West Midlands region of the UK, all patients with a suspected myeloproliferative neoplasm (MPN) have access to quantitative analysis of JAK2 V617F by droplet digital PCR as standard of care. The British Committee for Standards in Haematology recommends that suspected MPN cases have investigation of JAK2 exon 12, CALR and MPL genes if JAK2 V617F is negative.
Aims
The aim of the project was to improve the MPN service by substituting sequential analysis of individual target regions within the JAK2, CALR and MPL genes with a single assay, and to increase the number of genes available for analysis.
Methods
A commercial next generation sequencing (NGS) gene panel (Oxford Gene Technology, SureSeq Myeloid Panel), coupled with the Illumina MiSeq platform was validated and implemented. The gene panel utilises hybridization based enrichment technology and consists of 25 MPN–related genes. During the validation stage the following were enriched and analysed: 29 positive control samples with 30 known pathogenic variants, 30 negative control samples without known pathogenic variants in the JAK2, CALR and MPL genes, and 24 MPN samples of unknown mutational status. Thus so far over 1500 clinical samples have been analysed and reported since the service was introduced in October 2016.
Results
The panel has successfully identified: a large range of known pathogenic variants at high sensitivity (JAK2 V617F variant allele frequency 1%, CALR Type I frameshift variant allele frequency 3%), a potential alternative driver mutation in a known low level JAK2 V617F positive patient, a rare MPL exon 4 pathogenic variant and also the detection of low level CALR pathogenic variants, which would not have been detected by Sanger sequencing analysis. In one patient the panel identified the presence of two different JAK2 exon 14 pathogenic variants in cis (JAK2 V617F and JAK2 C618R). The JAK2 C618R prevented the hybridization of the probe binding site of the JAK2 V617F ddPCR assay which had led to a false negative result by ddPCR. The validation procedure also explored coverage and limits of sensitivity, potential chemistry specific artefacts and identified common polymorphisms for all 25 genes.
Conclusion
The panel has replaced the current sequential analysis of CALR, MPL and JAK2 exon 12 in JAK2 V617F negative patients and reduced turn-around-times with increased accuracy and sensitivity compared to Sanger sequencing and fragment analysis. Our current clinical service operates on a two tier system whereby clinicians can request analysis of the full 25 gene panel or a 4 gene subset (JAK2, CALR, MPL, CBL as an in silico analysis).
Session topic: 16. Myeloproliferative neoplasms - Clinical
Keyword(s): mutation analysis, Myeloproliferative disorder