Immunoglobulin Heavy Chain Variable Region (IGHV) Somatic Hypermutation Analysis of Bitypic CLL Cases Detects Prognostically Different Clones with Different Variable Region Segment Usage
Joshua F Coleman, Gwyneth K Olson, James Gale, Kristin E Hunt, Mohammad A Vasef. University of New Mexico/TriCore Reference Laboratories, Albuquerque, NM; Henry Ford Hospital, Detroit, MI
Background: Chronic lymphocytic leukemia (CLL) is a prognostically heterogeneous disease with survival based on the status of CD38 expression, cytogenetic abnormalities and IGHV somatic hypermutation. Nearly all CLL cases demonstrate a distinct flow cytometric immunophenotype including surface immunoglobulin light chain restriction and co-expression of CD5 and CD23. However, rare cases of CLL demonstrate bitypic light chain expression mimicking a polyclonal pattern when a limited panel of antibodies is used. Based on isolated reports, bitypic CLL appears to be biclonal, assessed by Southern blot and/or PCR analysis of IGH@ rearrangements. To the best of our knowledge, only 3 such bitypic, biclonal cases have been reported in the English literature. Somatic hypermutation status has not been previously analyzed.
Design: Six patients with bitypic light chains and an immunophenotype characteristic of CLL were identified from the archives at the University of New Mexico between 1998 and 2011. Hypermutation status was analyzed in 3 out of 6 cases in which DNA was available. Briefly, extracted DNA was subjected to PCR amplification using multiple primer sets flanking framework 1, 2, 3, and leader regions of IGHV. Following separation by capillary electrophoresis and Sanger sequencing, sequences were aligned with the ImMunoGeneTics database to determine IGHV segment usage and percent homology. Cases with less than 98% homology were designated as hypermutated.
Results: In 2 of the 3 cases with hypermutation analysis, 2 concurrent clones were confirmed by differential IGHV segment usage. The first case demonstrated hypermutated sequences with IGHV4-34, IGHV5-51, and IGHV3-30 usage. The second case revealed a hypermutated sequence with IGHV4-34 usage and an unmutated sequence with IGHV3-21. Analysis of the 3rd case was not possible due to mixed sequences, secondary to close proximity of amplicon sizes.
Conclusions: Bitypic CLL is rare but may be more common than previously described. By mimicking a polyclonal light chain pattern, such cases may be missed by limited flow cytometry screening panels. While inclusion of markers typically co-expressed in CLL (e.g., CD5 and CD23) aids in the correct interpretation, biclonality is confirmed by molecular studies. Analysis of hypermutation status can potentially distinguish between clones and furthermore determine whether either or both clones demonstrate an unfavorable unmutated genotype.
Monday, March 19, 2012 1:00 PM
Poster Session II # 199, Monday Afternoon