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HelpTest Code BCLL IGH Somatic Hypermutation Analysis, B-Cell Chronic Lymphocytic Leukemia (B-CLL), Varies
Reporting Name
IGH Somatic Hypermutation in B-CLLUseful For
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.
Method Name
Polymerase Chain Reaction (PCR) and Next-Generation Sequencing (NGS)
Performing Laboratory
Mayo Clinic Laboratories in Rochester
Specimen Type
VariesShipping Instructions
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 is required.. 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, complete blood cell count results and relevant clinical notes), and clinical or morphologic suspicion.
Specimen Required
Submit only 1 of the following specimens:
Specimen Type: Whole blood
Container/Tube:
Preferred: Lavender top (EDTA)
Acceptable: Yellow top (ACD)
Specimen Volume: 4 mL
Collection Instructions:
1. Invert several times to mix blood.
2. Send whole blood specimen in original tube. Do not aliquot.
3. Label specimen as blood.
Specimen Stability: Refrigerated/Ambient
Specimen Type: Bone marrow
Container/Tube:
Preferred: Lavender top (EDTA)
Acceptable: Yellow top (ACD)
Specimen Volume: 2 mL
Collection Instructions:
1. Invert several times to mix bone marrow.
2. Send bone marrow specimen in original tube. Do not aliquot.
3. Label specimen as bone marrow.
Specimen Stability: Refrigerated/Ambient
Specimen Type: Extracted DNA from blood or bone marrow
Container/Tube: 1.5- to 2-mL screw-top tube
Specimen Volume: Entire specimen
Collection Instructions:
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
Specimen Minimum Volume
Blood/Bone marrow: 1 mL
Extracted DNA: see Specimen Required
Specimen Stability Information
| Specimen Type | Temperature | Time | Special Container |
|---|---|---|---|
| Varies | Varies | 7 days |
Reject Due To
| Gross hemolysis | Reject |
| Moderately to severely clotted | Reject |
Special Instructions
Reference Values
An interpretive report will be provided.
Day(s) Performed
Monday, Wednesday, Friday
CPT Code Information
81263-IGH (immunoglobulin heavy chain locus) (eg, leukemia and lymphoma, B-cell), variable region somatic mutation analysis
LOINC Code Information
| Test ID | Test Order Name | Order LOINC Value |
|---|---|---|
| BCLL | IGH Somatic Hypermutation in B-CLL | 50627-9 |
| Result ID | Test Result Name | Result LOINC Value |
|---|---|---|
| 39465 | BCLL Result | No LOINC Needed |
| MP005 | Specimen Type | 31208-2 |
| 19674 | Final Diagnosis | 50398-7 |
Clinical Information
During early B-cell development, IGH genes are assembled from multiple polymorphic gene segments that undergo rearrangements and selection, generating variable diversity joining (VDJ) combinations that are unique in both length and sequence for each B cell. In addition, newly acquired (somatic) point variations are introduced into the variable (V) regions of mature B cells during the germinal center reaction in lymph nodes in a process called somatic hypermutation (SHM). Since chronic lymphocytic leukemia (CLL) originates from the malignant transformation of single lymphoid cells, each daughter cell shares one or, sometimes, more unique "clonal" antigen receptor gene rearrangements, which are cell and tumor specific (ie, a tumor cell "fingerprint"). Clonal IGHV gene hypermutation status provides important prognostic information for patients with CLL and small lymphocytic lymphoma (SLL). The presence of IGH SHM is defined as greater than 2% difference from the germline VH gene sequence identity (mutated), whereas less than or equal to 2% difference is considered no SHM (unmutated). The status of SHM has clear influence on the median survival of CLL patients. Hypermutation of the IGH variable region is strongly predictive of a good prognosis, while lack of variants predicts a poorer prognosis.
Interpretation
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).
Cautions
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.
Method Description
DNA is extracted from whole blood or bone marrow specimens, and IGH gene rearrangements are amplified by polymerase chain reaction (PCR) using indexed leader and/or framework region 1 (FR1) forward primers. The amplified product is then purified, and the DNA concentration measured. Pooled patient samples (identifiable by the index bar codes) are subjected to next-generation sequencing. FASTQC sequence data is subsequently analyzed using proprietary software to identify the IGH V rearrangement and the unique sequence. Results are compared to a germline IGHV sequence database by the software to calculate the percent identity of the tumor IGH V rearrangement to the closest germline sequence. Rearrangements containing a variation frequency of greater than 2% are interpreted as mutated. Rearrangements containing a variation frequency less than or equal to 2% are interpreted as unmutated.(Unpublished Mayo method)