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IgVH Somatic Hypermutation by Next Generation Sequencing

Test Code

M IGVH

Associations

Chronic Lymphocytic Leukemia, CLL / SLL

Reflex testing
Can be ordered as a reflex or in place of ZAP-70 by flow cytometry
Methodology

Next Generation Sequencing

Turnaround Time

10-14 days

Specimen Requirements

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

Specimen Stability
EDTA whole blood or bone marrow stable at room temperature (18-25°C)
Storage & Handling

Whole blood and bone marrow ship in a Styrofoam container with a cool pack. (Do not allow cool/refrigerated pack to directly contact samples)

Causes for Rejection

Insufficient volume; Specimen at incorrect temperature; Incorrect anticoagulant

Reference Range

(% homology to reference germline IGH sequence)

  • <97% = POSITIVE
  • 97-98% = Borderline
  • >98% = NEGATIVE
Description

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.

References

Read more about IgVH Somatic Hypermutation testing at MPLN here.

  1. International CLL-IPI working group.  An international prognostic index for patients with chronic lymphocytic leukaemia (CLL-IPI): a meta-analysis of individual patient data.  Lancet Oncol. 2016 Jun;17(6):779-90.
  2. Duncavage et al, Template for Reporting Results of Biomarker Testing of Specimens From Patients With Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma.  Arch Pathol Lab Med. 2016 Apr 15.
  3. Wilhelm C, Neubauer A, Brendel C. 2006. Discordant results of flow cytometric ZAP-70 expression status in B-CLL samples if different gating strategies are applied.  Cytometry B Clin Cytom. 2006 Jul 15;70(4):242-50.
  4. Sheikholeslami MR et al.  Variations in the detection of ZAP-70 in chronic lymphocytic leukemia: Comparison with IgV(H) mutation analysis.  Cytometry B Clin Cytom. 2006 Jul 15;70(4):270-5.
  5. Szankasi P, Bahler DW.  Clinical laboratory analysis of immunoglobulin heavy chain variable region genes for chronic lymphocytic leukemia prognosis.  J Mol Diagn. 2010 Mar;12(2):244-9.
  6. Rassenti et al.  ZAP-70 compared with immunoglobulin heavy-chain gene mutation status as a predictor of disease progression in chronic lymphocytic leukemia.  N Engl J Med. 2004 Aug 26;351(9):893-901.