Chronic Lymphocytic Leukemia, CLL / SLL
Next Generation Sequencing
5.0 mL (min. 1.0 mL) whole blood EDTA preferred, heparin whole blood accepted
3.0 ml (min. 1mL) bone marrow aspirate EDTA preferred, heparin accepted
Whole blood and bone marrow ship in a Styrofoam container with a cool pack. (Do not allow cool/refrigerated pack to directly contact samples)
Insufficient volume; Specimen at incorrect temperature; Incorrect anticoagulant
(% homology to reference germline IGH sequence)
CLL/SLL is the most common leukemia diagnosed among adults in Western countries and is associated with heterogeneous clinical outcomes. IgVH somatic hypermutation (SHM) status is a primary component of the CLL International Prognostic Index (CLL-IPI) working group formulation for disease risk stratification (1). Un-mutated IgVH has been established as a strong and independent predictor of adverse clinical prognosis and reduced overall survival.
Traditionally, IgVH SHM status has been evaluated by rt-PCR followed by Sanger Sequencing using RNA extracted from patient samples, however lability of RNA starting material places significant burden on the laboratory and submitting physician to ensure specimen transit time is minimized. Furthermore, sensitivity of IgVH SHM detection may be limited for low abundance CLL/SLL clones using traditional approaches, and Sanger Sequencing is also relatively time and labor intensive. Given these and other challenges, ZAP-70 expression by flow cytometry has been widely utilized as a surrogate marker for IgVH SHM status, where positive ZAP-70 expression is usually associated with absence of IgVH SHM and an unfavorable clinical prognosis (6). Unfortunately, standardization for this marker is known to be poor. Interpretation of the flow cytometry list mode data may be highly subjective, contributing to significant interobserver and interlaboratory variability in clinical reporting (3). Significant discordances between ZAP-70 expression patterns and expected IgVH SHM results have also been reported and attributed in some studies to pre-analytic sample processing factors (4)
Using a Next Generation Sequencing (NGS) based approach to IgVH SHM detection, laboratory workflow and data interpretation are relatively streamlined and automated, resulting in non-subjective data output that includes relative frequencies of clonal IGH digital sequence reads and their respective VDJ gene utilization profiles. The MPLN NGS methodology also utilizes DNA extracted from patient samples, eliminating the challenges related to RNA based testing.