Molecular diagnostics for AML
The AML Panel (AML.20141205), request code P042, exists of 327 amplicons and is in silico covering 100% of submitted areas (all coding regions (exons)) and is able to analyze variants in 19 genes implicated in AML.
Indicated exons (Table 1) include flanking intronic regions based on 5 base exon padding. For some genes the 5 base exon padding is not achieved or only a hotspot location is covered. See Table 2 for detailed coverage information about these aberrant regions.
Design AML Panel
Indicated exons include flanking intronic regions based on 5 base exon padding. For some genes the 5 base exon padding is not achieved or only a hotspot location is covered. See table 2 for detailed coverage information.
Table 1. Design AML panel
|
Gene |
Chromosome |
NCBI Transcript |
Exon |
Coverage |
|
ASXL1 |
Chr20 |
NM_015338.5 |
13 |
100% |
|
BRAF |
Chr7 |
NM_004333.4 |
15 |
100% |
|
CBL |
Chr11 |
NM_005188.3 |
8, 9 |
100% |
|
CEBPA |
Chr19 |
NM_004364.4 |
1 (full) |
100% |
|
DNMT3A |
Chr2 |
NM_022552.4 |
2-23 (full) |
100% |
|
FLT3 |
Chr13 |
NM_004119.2 |
16, 20, 21 |
100% |
|
GATA2 |
Chr3 |
NM_032638.4 |
3-7 (full) |
100% |
|
IDH1 |
Chr2 |
NM_005896.3 |
4 |
100% |
|
IDH2 |
Chr15 |
NM_002168.3 |
4 |
100% |
|
JAK2 |
Chr9 |
NM_004972.3 |
14 |
100% |
|
KIT |
Chr4 |
NM_000222.2 |
8, 10, 11, 17 |
100% |
|
KRAS |
Chr12 |
NM_033360.3 |
2, 3 |
100% |
|
NPM1 |
Chr5 |
NM_002520.6 |
11 |
100% |
|
NRAS |
Chr1 |
NM_002524.4 |
2, 3 |
100% |
|
PTPN11 |
Chr12 |
NM_002834.4 |
3, 13 |
100% |
|
RUNX1 |
Chr21 |
NM_001754.4 |
3-8 |
100% |
|
TET2 |
Chr4 |
NM_001127208.2 |
3-11 (full) |
100% |
|
TP53 |
Chr17 |
NM_000546.5 |
2-11 |
100% |
|
TP53β |
Chr17 |
NM_001126114.2 |
alt 10 |
100% |
|
TP53γ |
Chr17 |
NM_001126113.2 |
alt 10 |
100% |
|
WT1 |
Chr11 |
NM_024426.4 |
7, 10 |
100% |
Table 2. Aberrant covered regions AML panel
|
Gene |
Exon |
Coding DNA region |
|
BRAF |
15 |
c.1763 - c.1852 |
|
FLT3 |
16 |
c.1995 - c.2053+5 |
|
FLT3 |
20 |
c.2430 - c.2541+5 |
|
FLT3 |
21 |
c.2542-5 - c.2653+2 |
|
TP53 |
2 |
c.-5 - c.74+2 |
|
TP53 |
3 |
c.75-5 - c.96+2 |
Coverage of the NGS AML Panel
Coverage is the number of times a base is sequenced. The deeper the coverage of each base the greater the reliability and sensitivity of the sequencing assay. The minimum depth of coverage required for detection of somatic variants with the AML AmpliSeq Panel is 500X. The percentage of Target Base coverage (%Base500x) is the percentage of target bases in a panel that is covered at least 500 times.
The percentage of target bases that is covered at least 500 times (%Base500x) is at least 99.38% at 2.500.000 Mapped Reads. With this acceptance criteria two amplicons failed to yield >500 times coverage, specific locations are listed in Table 3. One amplicon in DNMT3A results in a too low coverage of 80bp at the 3’side of exon 4. For exon 4 one variant c.226C>T (p.P76S) is described in the COSMIC database. The mutation was encountered in 1 out of 125 (0.8%) examined samples and was related to an ovary tumor. Besides DNMT3a, one amplicon in RUNX1 has less than 500x coverage of 72bp in exon 4 and 5bp in intron 3. There are several AML related variants described in this region.
Table 3. Information failed amplicons AML panel
|
|
GRCh37/hg19 coordinates |
|
|
|
|
|
|
Chr |
Start |
End |
Gene |
Missing bases |
Exon |
Missing COSMIC |
|
Chr2 |
25505496 |
25505600 |
DNMT3A |
80 |
4 |
c.226C>T (p.P76S) |
|
Chr21 |
36259322 |
36259413 |
RUNX1 |
77 |
4 |
Several |
Design AML Fusion Transcript Panel
The AML-Fusion Transcript Panel (IAD84643) is able to detect fusion transcripts BCR::ABL1, CBFB::MYH11, PML::RARA and RUNX1::RUNX1T1 (AML1::ETO).
Fusion of genes may result in the usage of different donor and/or acceptor exons. Consequently, the corresponding fusion gene transcripts will have different exon compositions. In Table 4, a summation is listed with the number of fusion forms that are detected by the AML Fusion Transcript Panel.
Table 4. Overview fusion transcripts in AML Fusion Transcript Panel
|
Fusion Gene |
Transcript Variants |
|
BCR::ABL1 |
18 |
|
CBFB::MYH11 |
11 |
|
PML::RARA |
5 |
|
RUNX1::RUNX1T1 |
5 |
Reporting: addition hematological malignancies variants
This test does not distinguish between somatic and germ line alterations in analyzed gene regions, particularly when variant allele frequencies (VAF) are near 50% or 100%. If nucleotide alterations in genes associated with germ line mutation syndromes are present and there is also a strong clinical suspicion or family history of malignant disease predisposition, appropriate genetic counselling may be indicated.
Variants detected between 5% and 10% Variant Allele Frequency may indicate subclonal tumor populations. However the clinical significance of these findings may not always be distinct. It is demonstrated that in blood DNA samples from individuals with advancing age and who do not have a hematologic neoplasm, a low incidence of gene variants that are associated with myeloid neoplasms can be detected. This phenomenon of clonal hematopoiesis of indeterminate potential (CHIP) may not be clearly distinguishable from tumor-associated mutations, especially if detected as a sole abnormality (Steensma et al).
Correlation with clinical, histopathologic and additional laboratory findings is required for final interpretation of the results. The final interpretation of results for clinical management of the patient is the responsibility of the managing physician.
Service pages NGS
