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Providing prognostic information in patients with newly diagnosed B-cell chronic lymphocytic leukemia
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 or equal to 2% difference from the germline VH gene sequence identity (mutated), whereas less than 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.
An interpretive report will be provided.
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 2% or greater 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).
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.
The 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.
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