Overview
This custom NGS panel is an amplicon-based targeted sequencing assay designed to detect recurrent somatic DNA variations (SNVs and small indels) in genes that are clinically relevant to chronic lymphocytic leukemia (CLL).
Indications
The CLL NGS panel is offered to patients with a confirmed diagnosis of CLL to aid in treatment decisions and is not indicated for diagnostic purposes. This testing should be performed prior to the initiation of treatment, rather than at the time of diagnosis. Repeat testing upon disease progression while on treatment may be indicated. See here for information on CLL FISH testing.
Test Requirements
For peripheral blood (preferred specimen)
- Peripheral blood (DNA): 1 x 6ml EDTA (NGS Panel)
For bone marrow
- Bone marrow (DNA): 1 x 0.5ml in EDTA tube (NGS Panel)
For FFPE Tissue Block (NGS Panel ONLY)
- FFPE tissue block: will be sectioned
- Specimen must not be de-calcified
- Minimum tumour content is 10-20%
Completed CGL Lymphoid Requisition form.
See sample Specimen Guidelines for additional information for acceptable sample types.
Turn around time
Results are reported within 21 days from receipt of the necessary specimen, completed requisition form and all relevant documentation.
Results Reporting
Identified variants are classified into tiers of clinical significance:
- TIER I – VARIANTS OF STRONG CLINICAL SIGNIFICANCE
- TIER II – VARIANTS OF POTENTIAL CLINICAL SIGNIFICANCE
- TIER IIIA –VARIANTS OF UNCERTAIN CLINICAL SIGNIFICANCE
- TIER IIIB – VARIANTS OF UNCERTAIN FUNCTION
Please see our Variant Classification Guidelines for additional details.
Genes targeted
Either the coding exonic sequence and at least 5 bp of flanking intronic sequence, or known hotspots of clinical importance, are assessed in the following genes.
Reporting of variants in these genes is limited to Tiers I, II, IIIA and IIIB: Entire coding region (+/- a minimum of 5 bp of adjacent introns): BCL2:NM_000633; PLCG2: NM_002661; TP53:NM_000546.6; Partial coding region (+/- a minimum of 5 bp of adjacent introns): BIRC3:NM_001165 (exons 4 to 9); BTK:NM_000061 (exons 14 to 16); MYD88: NM_002468.5 (exon 5); NOTCH1: NM_017617 (exon 34); SF3B1: NM_012433 (exons 14 to 16).
Methods
The test is performed on genomic DNA extracted from the submitted specimen (as above) using the Maxwell RSC instrument. Targeted DNA regions are amplified by multiplex PCR. Paired-end massively parallel sequencing of 300-bp fragments is performed with an illumina MiSeq i100 instrument. DNA sequences are aligned and compared to reference genome hg38/GRCh38 to identify single nucleotide variants (SNVs) and small insertions and deletions (indels). Variants are interpreted and categorized based on their clinical impact using AMP/ASCO/CAP guidelines (PMID: 27993330) as follows: Tier I, variants with strong clinical significance (level A and B evidence); Tier II, variants with potential clinical significance (level C and D evidence); Tier IIIA, variants with uncertain clinical significance; Tier IIIB, variants with uncertain function and Tier IV, (likely) benign variants. Reporting of Tier IIIB variants may be context dependent. Tier IV variants are not reported. Unless otherwise noted, variant allele frequencies are obtained from The AACR Project GENIE Consortium (PMID: 28572459, mycancergenome.org).
References
- Malcikova J, et al. (2018) ERIC recommendations for TP53 mutation analysis in chronic lymphocytic leukemia – update on methodological approaches and results interpretation. Leukemia PMID: 29467486.
- Campo E, et al. (2018) TP53 aberrations in chronic lymphocytic leukemia: an overview of the clinical implications of improved diagnostics. Haematologica PMID: 30442727
- Zenz T, et al. (2010) TP53 mutation and survival in chronic lymphocytic leukemia. J Clin Oncol. PMID: 20697090.