Abstract

Chronic lymphocytic leukemia (CLL) is characterized by a heterogeneous clinical course where some patients have an indolent disease and survive for decades while others have a rapidly progressive disease with short survival. Clinical staging according to Rai or Binet are useful in predicting survival. However, in early stages a subset of patients progress while others remain stable. CLL can be subdivided into two groups based on IgVH gene mutation status, and it has been shown that patients with unmutated IgVH genes have poorer survival irrespective of clinical stage. A major drawback using mutation status as a prognostic marker is that it is expensive and time consuming. Therefore, there is a need for surrogate markers that can be analyzed in routine laboratories.

We analyzed the expression of CD69, CD38 and ZAP-70 in CD19+ cells from CLL patients (n=28) and healthy donors (n=10) using flow cytometry. The gene expression of ZAP-70 was also analyzed by real-time PCR. CD69 was higher expressed in B cells from CLL patients compared with controls (35±31% and 2.6±1.8% CD69+/CD19+ cells respectively, P=0.001). Patients with unmutated IgVH genes (UM CLL) had a higher percentage of CD69+ cells compared with patients with mutatated IgVH (M CLL; 70±24% vs. 18±12%, P=0.00076). Furthermore, there was a strong concordance, even better than CD38 and ZAP-70, between IgVH mutation status and expression of CD69 (96%). Thus, we suggest that CD69 expression can be used as a surrogate marker for mutation status. Furthermore, this analysis is reliable and inexpensive.

Figure 1.

A) Flow cytometry of CD69 in CD19+ cells in one representative control, mutated and unmutated CLL patient, respectively. B) CD19+ B cells from 10 controls and 28 patients were analyzed for CD69 expression. % CD69+/CD19+ cells in controls vs. all CLL patients, Binet A vs. Binet B+C and mutated vs. unmutated CLL patient. Horizontal lines represent mean values in each group. C) Concordance between IgVH mutation status and CD69, CD38 and ZAP-70. Individuals with ≥ 20% positive CD19+ cells were defined as positive for CD38 and ZAP-70. For CD69, individuals with ≥ 50% positive CD19+ cells were defined as “high CD69” and individuals with <50% CD69 were defined as “low CD69”. Percentage deviation from the most similar germline gene is shown. Genes with ≥ 2% deviation (dotted line) were defined as mutated. D) ZAP-70 mRNA expression was analyzed in 22 patients by RT-PCR. Mean ZAP-70/RPLP0 expression±SD in CLL patients with mutated IgVH genes (n=16) vs. unmutated IgVH genes (n=6) are shown.

Figure 1.

A) Flow cytometry of CD69 in CD19+ cells in one representative control, mutated and unmutated CLL patient, respectively. B) CD19+ B cells from 10 controls and 28 patients were analyzed for CD69 expression. % CD69+/CD19+ cells in controls vs. all CLL patients, Binet A vs. Binet B+C and mutated vs. unmutated CLL patient. Horizontal lines represent mean values in each group. C) Concordance between IgVH mutation status and CD69, CD38 and ZAP-70. Individuals with ≥ 20% positive CD19+ cells were defined as positive for CD38 and ZAP-70. For CD69, individuals with ≥ 50% positive CD19+ cells were defined as “high CD69” and individuals with <50% CD69 were defined as “low CD69”. Percentage deviation from the most similar germline gene is shown. Genes with ≥ 2% deviation (dotted line) were defined as mutated. D) ZAP-70 mRNA expression was analyzed in 22 patients by RT-PCR. Mean ZAP-70/RPLP0 expression±SD in CLL patients with mutated IgVH genes (n=16) vs. unmutated IgVH genes (n=6) are shown.

Disclosures: No relevant conflicts of interest to declare.

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