Background: The mutational status of immunoglobulin VH genes (IgVH) is an important prognostic marker in chronic lymphocytic leukemia (CLL), but its determination remains unadapted to routine practice. Several reports have showed that ZAP-70, whose expression can be detected by flow cytometry, can be considered as a reliable surrogate marker of IgVH mutational status. We recently conducted a gene expression profiling study of 18 cases of CLL, which pointed out 2 other genes which might also discriminate CLL groups: the lipoprotein lipase (LPL) and the disintegrin and metalloprotease 29 (ADAM29) genes which were overexpressed preferentially in unmutated (UM) and mutated (MT) cases respectively.
Methods: We analyzed frozen cells obtained at diagnosis for 127 CLL patients (87 Binet stage A, 40 stage B or C). LPL, ADAM29 and house keeping GAPDH gene expression were measured by real time quantitative polymerase chain reaction in 111 cases, and ZAP-70 protein by flow cytometry in 107 cases, both analyses being performed in 93 cases. LPL and ADAM29 were also quantified in peripheral blood mononuclear cells (PBMC, n=4) and purified B cells (n=3) of healthy individuals. We correlated the results with the previously determined IgVH mutational status and clinical outcome.
Results: With a cut-off value determined to be 1 for the LPL/GAPDH copy number ratio, LPL expression had a positive predictive value (PPV) of 68% for UM cases and a negative predictive value (NPV) of 85% for MT patients. Alternatively ADAM29 expression (ADAM29/GAPDH > 3) had a PPV of 77% for MT cases and of 86% for UM cases. Combining LPL and ADAM29 RNA quantifications by a simple 1:1 ratio (L/A ratio; threshold=1) provided slightly better results than those obtained with ZAP-70 (positivity threshold = 20%) for PPV of UM status (90% vs 76%) and similar results for NPV of MT status (90% vs 91%). Simultaneaous usage of L/A ratio and ZAP-70 expression allowed an almost perfect (73/74) assessment of the IgVH status in 80% (74/93) of patients with concordant results (L/A+, ZAP-70+ or L/A-, ZAP-70-). Serial measurements of L/A ratio and ZAP-70 expression showed that both parameters can change over time (median follow-up 38 months, range 6–159) in a fraction of patients (5/25 tested). ADAM29 was not detected while LPL was present at very low levels or absent in PMBC or purified B cells from healthy donors. The IgVH mutational status, ZAP-70 and L/A ratio were predictive of event-free survival for the whole cohort and for stage A patients. However only the L/A ratio was significantly associated with a shorter survival (p=0.03) for stage B/C patients.
Conclusions: Combination of LPL and ADAM29 mRNA quantification provides a good surrogate marker of the IgVH mutational status in CLL patients. Used in association with ZAP-70, it represents an reliable alternative to the sequencing of IgVH genes. In addition, it might constitute a more powerful prognostic marker than IgVH mutational status or ZAP-70.