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Test Code BCLL IGH Somatic Hypermutation Analysis, B-Cell Chronic Lymphocytic Leukemia (B-CLL)

Useful For

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

Reporting Name

IGH Somatic Hypermutation in B-CLL

Specimen Type

Varies


Shipping Instructions


1. Specimen must arrive within 3 days (72 hours) of draw.

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



Necessary Information


The following information is required:

1. Pertinent clinical history

2. Clinical or morphologic suspicion

3. Date of collection

4. Specimen source



Specimen Required


Submit only 1 of the following specimens:

 

Specimen Type: Peripheral blood

Container/Tube:

Preferred: EDTA (lavender top)

Acceptable: ACD (yellow top)

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 Type: Bone marrow

Container/Tube:

Preferred: EDTA (lavender top)

Acceptable: ACD (yellow top)

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 Information

Specimen Type Temperature Time
Varies Refrigerated (preferred) 72 hours
  Ambient  72 hours

Reject Due To

Hemolysis

Mild OK; Gross reject

Lipemia

NA

Icterus

NA

Other

Moderately to severely clotted

Clinical Information

During early B-cell development, IGH genes are assembled from multiple polymorphic gene segments that undergo rearrangements and selection, generating VDJ combinations that are unique in both length and sequence for each B cell. In addition, new acquired (somatic) point mutations 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 mutation predicts a poorer prognosis. Although the determination of mutation 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 (eg, using the Illumina MiSeq platform) 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 mutation percent.

Reference Values

An interpretive report will be provided.

Interpretation

The presence or absence of somatic hypermutation in the immunoglobulin heavy chain gene (IGH) variable (V) region DNA will be reported. A mutation 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 somatic hypermutation of the IGHV region (unmutated) is associated with a significantly worse prognosis than B-CLL containing somatic hypermutation of the IGHV 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 IGHV mutation status is applicable at this time only to this subtype of B-cell malignancy and the test is not intended for use in other B-cell neoplasms or hematopoietic tumors.

 

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, et al: Unmutated Ig V(H) genes are associated with a more aggressive form of chronic lymphocytic leukemia. Blood 1999 September 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 September 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;25:979-984

Method Description

RNA is extracted from B-cell chronic lymphocytic leukemia specimens and converted to cDNA using reverse transcription. PCR is then used to amplify the IGH gene rearrangements with indexed multiplex primers designed to include a portion of the leader segment, all of the variable (V) and diversity (D) segments, and a portion of the joining (J) segment. A check gel is performed to make sure that there is amplified product. If no amplified product is visible a second PCR is performed with a different multiplex primer set targeting the framework 1 (FR1) regions of the V genes. FR1 PCR rearrangements are shorter in length than leader primer products and result in slightly truncated 5' V-region sequence coverage. The amplified product is then purified and the DNA concentration measured. Pooled patient samples (identifiable by the index bar codes) are subjected to sequencing on the Illumina-MiSeq platform. FASTQC sequence data is subsequently analyzed using proprietary software to identify the IGHV rearrangement and the unique sequence. Results are compared to a germline IGVH sequence database by the software to calculate the percent identity of the tumor IGHV rearrangement to the closest germline sequence. Rearrangements containing a mutation frequency of greater than 2% are interpreted as mutated. Rearrangements containing a mutation frequency less than or equal to 2% are interpreted as unmutated.(Warren EH, Matsen IV FA, Chou J: High-throughput sequencing of B- and T-lymphocyte antigen receptors in hematology. Blood 2013 Jul 4;122(1):19-22; Schumacher JA, Duncavage EJ, Mosbruger TL, et al: A Comparison of Deep Sequencing of TCRG Rearrangements vs Traditional Capillary Electrophoresis for Assessment of Clonality in T-Cell Lymphoproliferative Disorders. Am J Clin Pathol 2014 Mar;141(3):348-359; Blachly JS, Ruppert AS, Zhao W: Immunoglobulin transcript sequence and somatic Hypermutation computation from unselected RNA-seq reads in chronic lymphocytic leukemia. Proc Natl Acad Sci USA 2015;112(14):4322-4327)

Day(s) and Time(s) Performed

Wednesday, Friday

Analytic Time

Up to 2 weeks

Specimen Retention Time

Until reported

Performing Laboratory

Mayo Medical 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 U.S. 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 In Process

 

Result ID Test Result Name Result LOINC Value
39465 BCLL Result In Process
MP005 Specimen Type 31208-2
19674 Final Diagnosis 34574-4

Method Name

Polymerase Chain Reaction (PCR) and Next-Generation Sequencing

Specimen Minimum Volume

Blood or Bone Marrow: 1 mL

Forms

1. Molecular Hematopathology Patient Information: B-Cell Chronic Lymphocytic Leukemia (CLL) for IGVH and/or TP53 Somatic Mutation Testing in Special Instructions

2. If not ordering electronically, complete, print, and send a Hematopathology/Cytogenetics Test Request Form (T726) with the specimen (http://www.mayomedicallaboratories.com/it-mmfiles/hematopathology-request-form.pdf)