26208 B-Cell IGH Hypermutation Analysis (BCLL)

​BCLL
IGVH
IGHV
Next Gen Sequencing Test
Somatic mutation (or Hypermutation)
CLL prognosis

​Providing prognostic information in patients with newly diagnosed B-cell chronic lymphocytic leukemia

This test is not intended for use in providing prognostic information for patient with other B-cell neoplasms or hematopoietic tumors.

1. Both refrigerated and ambient specimens must arrive within 7 days of collection.

2. Collect and package specimen as close to shipping time as possible.

 Necessary Information:

1. Molecular Hematopathology Patient Information: B-Cell Chronic Lymphocytic Leukemia (CLL) for IGVH and/or TP53 Somatic Mutation Testing (T711) is required, see Special Instructions. Testing may proceed without the patient information, however, it aids in providing a more thorough interpretation. Ordering providers are strongly encouraged to fill out the form and send with the specimen.

2. If form is not provided, include the following information with the test request: specimen source, pertinent clinical history (ie, CBC results and relevant clinical notes), and clinical or morphologic suspicion.

Specimen Type: Peripheral blood

1. Invert several times to mix blood.

2. Send specimen in original tube.

3. Label specimen as blood.

Specimen Stability: Refrigerated/ Ambient

Specimen Type: Bone marrow

1. Invert several times to mix bone marrow.

2. Send specimen in original tube.

3. Label specimen as bone marrow.

Specimen Stability: Refrigerated/ Ambient

Specimen Type: Extracted DNA from blood or bone marrow

1. Label specimen as extracted DNA and indicate specimen source (blood or bone marrow).

2. The required volume of DNA is 50 mcL at a concentration of 20 ng/mcL

3. Include volume and concentration on tube.

Specimen Stability: Frozen (preferred)/Refrigerated

​This test is useful for patients with chronic lymphocytic leukemia (CLL), or small lymphocytic lymphoma (SLL) with blood or bone marrow involvement. The prognostic value of somatic IGH V (IGHV) region mutation status is applicable only for this subtype of B-cell malignancy.

This test requires a minimum monoclonal CLL B-cell percentage in order to amplify the clonal IGH gene rearrangement. This level has been established at 5% of lymphocytes (eg, as determined by flow cytometric immunophenotyping). A CLL population below 5% will not have a reliable or reproducible clonal gene rearrangement and sequencing by next-generation sequencing to determine somatic mutation status will typically produce no results, or possibly a false-positive finding. Therefore, submitted CLL samples must have a minimum CLL monoclonal B-cell population of 5% of total lymphocytes.

The prognostic significance of somatic hypermutation (SHM) status is only known when a single functional IGH rearrangement is identified (ie, in frame junctional coding region with no predicted premature protein truncation). However, a variety of situations can occur, for which the clinical significance is unknown at this time. These can broadly be grouped into the following:

1. Greater than 1 functional rearrangement is identified, with discordant mutation status

2. Only nonfunctional rearrangements are identified

Rearrangements with mutation status at or near the 2% cutoff should be interpreted with caution for the purposes of prognosis, particularly if the entire IGHV sequence could not be sequenced due to the use of framework region 1 (FR1) V region primers. If such results are identified, an appropriate comment will be provided in the report.

The presence or absence of somatic hypermutation (SHM) in the immunoglobulin heavy chain gene (IGH) variable (V) region DNA will be reported. A variation frequency of greater than 2% will be reported as mutated. Both the percent mutation and the V region allele identified in the rearrangement will be included in the report.

B-cell chronic lymphocytic leukemia (B-CLL) lacking SHM of the IGH V region (unmutated) is associated with a significantly worse prognosis than B-CLL containing SHM of the IGH V region (mutated).