The comprehensive profiling of gene expression in CLL presented here provides a new molecular framework for understanding the etiology of this leukemia and the divergent clinical courses of these patients. Using genomic-scale gene expression profiling, we addressed a current controversy in CLL pathogenesis, namely whether this diagnosis comprises more than one disease entity. CLL patients have been subdivided based on the Ig mutational status of their leukemic cells (1
), but it was unclear whether these patients had molecularly distinct diseases. Our data demonstrate that all CLL patients share a characteristic gene expression signature in their leukemic cells. These findings support a model in which all cases of CLL have a common cell of origin and/or a common mechanism of malignant transformation. In this model, the CLL-specific gene expression signature might represent the gene expression signature of a common normal precursor cell or it might reflect the downstream gene expression consequences of a common oncogenic event. These findings are in contrast to the previous observation that DLBCL consists of two disease entities that did not have overlapping gene expression outside of genes involved in proliferation and in the host response to the tumor (6
Previously unsuspected features of CLL biology emerge from its gene expression profile, generating a wealth of hypotheses to guide future studies of this disease. CLL cells proliferate slowly in vivo, driven by unknown signals. Therefore, it is notable that Wnt-3 was highly, and selectively, expressed in CLL ( B). The Wnt gene family encodes secreted proteins that signal through cell surface receptors of the frizzled family to control development and mediate malignant transformation (15
). Intriguingly, another CLL signature gene, Ror1, encodes a receptor tyrosine kinase with an extracellular domain that resembles a Wnt interaction domain of frizzled (16
). Recently, Wnt-3 has been shown to promote proliferation of mouse bone marrow pro-B cells by initiating signaling events leading to transcriptional activation by LEF-1(17
). Thus, CLL cells may use an autocrine mechanism of proliferation that is used normally by B cell progenitors.
We nevertheless also found that the expression of hundreds of other genes correlated with the Ig mutational status in CLL, providing insights into the biological mechanisms that lead to the divergent clinical behaviors of CLL patients. The most differentially expressed gene between the CLL subtypes was ZAP-70, a critical kinase that transduces signals from the T cell antigen receptor, and is preferentially expressed in normal T lymphocytes (18
). Differential expression of ZAP-70 between CLL subtypes was therefore surprising since its expression in normal B cells has not been previously reported. However, by microarray analysis and RT-PCR analysis we found that ZAP-70 mRNA is highly expressed in some B lymphoma cell lines along with being differentially expressed by the CLL subtypes. A ZAP-70–related kinase, syk, transduces signals from the BCR (19
), raising the possibility that ZAP-70 might alter BCR signaling in CLL cells. Another CLL subtype distinction gene, Pak1, could contribute to the resistance of CLL cells to apoptosis by phosphorylating Bad and thereby preventing Bad from inhibiting BCL-2 (20
). FGFR1 is a receptor tyrosine kinase that can stimulate cellular proliferation after interaction with fibroblast growth factors. The higher expression of FGFR1 in Ig-unmutated CLL is intriguing given that CLL patients have elevated blood levels of basic fibroblast growth factor which can activate FGFR1 and block apoptosis in CLL (21
Intriguingly, CLL subtype distinction genes were enriched for genes that are modulated in expression during signaling of B cells through the BCR. One hypothesis raised by this observation is that the leukemic cells in Ig-unmutated CLL may have ongoing BCR signaling. Interestingly, the VH
repertoire usage in the Ig-unmutated and Ig-mutated CLL is distinct (1
) and the combinations of VH
, and JH
gene segments rearranged in CLL cells are not random (1
). These observations suggest that the surface Ig receptors of CLL cells may have specificity for unknown environmental or self-antigens. Indeed, CLL cells have been shown to frequently produce antibodies that bind classical autoantigens (25
). The gene expression profiling data presented in this report raise the possibility that Ig-unmutated CLL cells may be continuously stimulated in vivo by antigen, giving rise to a gene expression profile that is reminiscent of BCR signaling. Indeed, CLL cells from patients with progressive disease were more readily stimulated by BCR cross-linking to synthesize DNA than were CLL cells from patients with stable disease (28
). Although this study did not distinguish between Ig-unmutated and Ig-mutated CLL, the results are consistent with a differential ability of these subtypes to signal through the BCR. Alternatively, it is possible that Ig-unmutated CLL cells activate the same signaling pathways that are engaged during B cell activation as a result of genetic changes in the leukemic cells or by other pathological mechanisms.
An immediate clinical application of the present results would be in the differential molecular diagnosis of CLL. We demonstrated that as few as 1–3 genes could correctly assign patients to a CLL subtype with 100% accuracy. Thus, our results could be used to establish a quantitative RT-PCR test to diagnose the CLL subtypes and that would be easier to adopt clinically than DNA sequence analysis of Ig variable regions. Given the relatively benign course of Ig-mutated CLL, a simple diagnostic test based on gene expression would provide valuable prognostic information for CLL patients and could be used to guide treatment decisions.
Finally, our results suggest new therapeutic approaches to this currently incurable leukemia. First, the protein products of some of the CLL signature genes may present new targets for mAb therapy and for vaccine approaches to CLL. Second, the unexpected finding that B cell activation genes were upregulated in Ig-unmutated CLL patients suggests the intriguing possibility that signaling pathways downstream of the BCR may contribute to the more progressive clinical course of these patients. Thus, therapeutic targeting of these signaling pathways could specifically benefit those CLL patients that show gene expression evidence that these pathways are active.