Analysis of IGVH Gene Rearrangements in a Chronic Lymphocytic Leukemia Cohort from a Large US Reference Laboratory
Christopher N Thompson, Philippe Szankasi, David W Bahler, Todd W Kelley. University of Utah, Salt Lake City, UT; ARUP Laboratories, Salt Lake City, UT
Background: Chronic lymphocytic leukemia (CLL) is the most common leukemia of adults in the United States. Somatic mutation status of the immunoglobulin variable region heavy chain (IgVH) gene is an important predictor of clinical behavior. In this study, IgVH gene usage and mutation status was analyzed in a cohort of 454 CLL patients from the United States in a reference laboratory setting.
Design: Peripheral blood samples were analyzed from 454 patients with the clinical history of CLL. After preparing RNA from whole blood, random-primed cDNA was generated and then amplified using VH family-specific leader region primers and JH-region reverse primers. VH3-21 was specifically amplified using a matched leader primer. Sequencing was performed and the data was compared to a database of human immunoglobulin sequences. Cases with <98% homology to the closest matching gene segment were classified as mutated. Additionally, mean and median sequence homology was determined for each VH gene segment.
Results: A total of 461 clonal rearrangements using 43 unique IgVH gene segments were detected in 454 patients including 7 patients with 2 clones. 4 of 7 cases (57%) with 2 clones demonstrated usage of VH1-69 in one of the clones and 2 of 7 cases with 2 clones had the same pattern (VH1-69 with VH4-34, both with different mutational status). Overall, 55.5% of cases were classified as mutated. Biased usage of IgVH gene segments was observed with overrepresentation of VH1-69 (n=50 cases; 10.9%), VH4-34 (n=47 cases; 10.3%), VH3-30 (n=37; 8.1%), VH3-23 (n=31; 6.8%), VH3-7 (n=29; 6.3%), VH1-2 (n=23; 5.0%), VH3-33 (n=22; 4.8%) and VH3-21 (n=22; 4.8%). Many of the frequently used IgVH segments also demonstrated a clear bias with respect to mutation status. VH1-69 was almost always unmutated (median 100% homology), while VH-4-34 (median 94.7% homology), VH3-23 (median 96.2% homology) and VH3-7 (median 94.4% homology) were most commonly mutated.
Conclusions: Our data from a large, unique reference laboratory testing population supports previous studies showing biased IgVH usage in CLL. Our finding of 4.8% VH3-21 positive cases is significantly higher than that reported in other large series from the US and supports our method of using a specific VH3-21 matched leader primer which likely enhances the sensitivity of our testing method. Analysis of cases with two clonal rearrangements has not previously been reported. Finding two clonal rearrangements with differing mutational status may present problems in assigning prognosis.
Tuesday, March 20, 2012 1:00 PM
Platform Session: Section G, Tuesday Afternoon