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EPIC CODE: BCLL IGH Somatic Hypermutation Analysis, B-Cell Chronic Lymphocytic Leukemia (B-CLL), Varies

Useful 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.

Reporting Name

IGH Somatic Hypermutation in B-CLL

Specimen Type


Shipping 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: 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 Required

Submit only 1 of the following specimens:


Specimen Type: Peripheral blood


Preferred: Lavender top (EDTA)

Acceptable: Yellow top (ACD)

Specimen Volume: 4 mL

Collection Instructions:

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


Preferred: Lavender top (EDTA)

Acceptable: Yellow top (ACD)

Specimen Volume: 2 mL

Collection Instructions:

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

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 Stability Information

Specimen Type Temperature Time Special Container
Varies Varies 7 days

Reject Due To

Gross hemolysis Reject
Moderately to severely clotted Reject

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, new acquired (somatic) point variations are introduced into the variable (V) regions of mature B cells during the germinal center reaction in lymph nodes, and this process is called somatic hypermutation (SHM). Since chronic lymphocytic leukemia (CLL) originates from the malignant transformation of single lymphoid cells, each daughter cell shares 1 or (sometimes) more unique "clonal" antigen receptor gene rearrangements, which are cell and, therefore, 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.

Reference Values

An interpretive report will be provided.


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).


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.

Clinical Reference

1. Hamblin TJ, Davis Z, Gardiner A, Oscier DG, Stevenson FK: Unmutated Ig V(H) genes are associated with a more aggressive form of chronic lymphocytic leukemia. Blood. 1999 Sep 15;94(6)::1848-1854

2. Damle RN, Wasil T, Fais F, et al: Ig V gene mutation status and CD38 expression as novel prognostic indicators in chronic lymphocytic leukemia. Blood. 1999 Sep 15;94(6):1840-1847

3. Langerak AW, Davi F, Ghia P, et al: Immunoglobulin sequence analysis and prognostication in CLL: guidelines from the ERIC review board for reliable interpretation of problematic cases. Leukemia. 2011 Jun;25(6):979-984. doi: 10.1038/leu.2011.49

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 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 IGHV 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 IGHV 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)

Day(s) Performed

Monday, Wednesday, Friday

Report Available

14 days

Specimen Retention Time

DNA 3 months

Performing Laboratory

Mayo Clinic Laboratories in Rochester

Test Classification

This test was developed, and its performance characteristics determined by Mayo Clinic in a manner consistent with CLIA requirements. This test has not been cleared or approved by the US Food and Drug Administration.

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

NY State Approved


Method Name

Polymerase Chain Reaction (PCR) and Next-Generation Sequencing

Secondary ID


Specimen Minimum Volume

Blood: 1 mL
Bone Marrow: 1 mL
Extracted DNA: see Specimen Required


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

2. If not ordering electronically, complete, print, and send a Hematopathology/Cytogenetics Test Request (T726) with the specimen.


Although the determination of variant status can be accomplished by PCR followed by Sanger sequencing, this approach only allows for analysis of single samples at a time. Next-generation sequencing (NGS) technology represents a significant improvement over existing Sanger assays by allowing for batch sample analysis and simultaneous identification of clonal IGH rearrangement, the tumor-specific rearrangement sequence, and determination of somatic variant percent.