We have provided novel evidence for lineage plasticity in mature human neoplastic B lymphocytes. We showed for the first time that human B-cells carrying the t(14;18) could be reprogrammed into macrophages through a downregulation of the B-cell transcription factor PAX5. Most theories of hematopoietic cell differentiation have proposed that as cells differentiate fully, they become lineage committed. However, some clinical data have shown that two hematopoietic populations in the same patient may share identical genetic changes or abnormalities, raising the possibility tumors expressing the phenotype of one hematopoietic lineage might transdifferentiate into a genetically similar but phenotypically distinct tumor of a different lineage. For example, we previously reported examples of patients with lymphoblastic leukemias or lymphomas who also had histiocytic or dendritic cell tumors and demonstrated that the lymphoblastic neoplasm and the histiocytic/dendritic cell tumor from each patient carried identical genetic changes, indicating a clonal relationship between the two tumors. However, tumors of precursor cells display more lineage plasticity than mature lymphoid tumors. Reprogramming of mature human B-cells into cells of a different lineage has not previously been shown, although this has been accomplished in follicular hyperplasia. We subsequently assayed the phosphorylated and total protein levels of several murine models. We identified a series of human histiocytic/dendritic cell tumors that arose in patients with prior follicular lymphoma (FL). In each case the FL cells carried the BCL2/JH translocation, which causes upregulation of BCL2 blocking apoptosis in the neoplastic B-cells, as confirmed by fluoresence in situ hybridization analysis and analysis using the polymerase chain reaction technique. The subsequent histiocytic/dendritic cell tumors were shown to be clonally related to the underlying FL, carrying the identical BCL2/JH translocation and identical immunoglobulin heavy chain gene rearrangements confirmed by sequence analysis. We showed that transdifferentiation of the neoplastic B-cells occurred through downregulation of the B-cell transcription factor PAX5, and upregulation of transcription factors expressed in histiocytes and myeloid cells, such as CEBPbeta. This paper was published as a Plenary paper in Blood. This is the first instance in which mature human B-cells have been shown to exhibit lineage plasticity in vivo, and even in vitro. It provides insight into the capacity for lineage plasticity in human cells, and has implications for understanding the potential of human cells to be reprogrammed into stem cells, with altered capacity for differentiation into other cell lines. Thus, it has implications beyond the hematopoietic system, and has relevance for generation of human stem cells. In a separate and related study, we explored the mechanisms of cellular control and immune regulation in follicular lymphoma. We hypothesized that the cytokine microenvironment might play a role in follicular lymphoma by influencing tumoral signaling networks that regulate survival, proliferation, and differentiation. Evidence suggests factors in the lymph node microenvironment, related to tumor infiltrating T-cells, macrophages, and dendritic cells, play a role in the disease process. To address this question, we performed cytokine proteomic profiling of follicular lymphoma from diagnostic lymph node biopsies prior to therapy, and compared the results to cases of benign downstream intracellular signaling proteins. We generated proteomic cytokine profiles of FL (N=50) and follicular hyperplasia (FH; N=23). Ten cytokines were assayed using ultrasensitive multiplex elisas: IL-1beta, IL-2, IL-4, IL-5, IL-8, IL-10, IL-13, IL-12p70, TNF-alpha, and INF-gamma. Each cytokine showed overall lower protein concentrations in FL, with the exception of IL-4, which was nearly 5 times higher in FL than FH (p= 0.0054). Using reverse-phase protein microarrays (RPMA), we evaluated the activation state of several intracellular signaling proteins downstream of cytokine receptors. Basal Erk phosphorylation was approximately 4 times greater in FL than FH (p less than 0.0001) with similar findings for Mek; Stat-6 showed weak basal phosphorylation that was approximately twice as great in FL than in FH (p = 0.012). In conclusion, the FL microenvironment contains increased levels of IL-4, with prominent tumor basal phosphorylation of Erk. These findings suggest that IL-4, Erk, and possibly Stat-6 may play a role in the biology of FL and may serve as targets for future therapies. These findings shed light on potential cooperative mechanisms of disease in follicular lymphoma and have implications for treatment targets in follicular lymphoma. We postulate that there may be a relationship between the relatively high IL-4 levels in follicular lymphoma and the sustained basal activation of Erk that we also detected. FL cells are reported to upregulate the IL-4 receptor-alpha. Additionally, IL-4 has been demonstrated to be a potent stimulator of follicular lymphoma cell proliferation in vitro, and Erk is an established regulator of proliferation within the MAP kinase pathway. Basal activation of Erk in follicular lymphoma may reflect dysregulation of signal transduction networks controlling proliferation, possibly via attenuated or lack of appropriate negative feedback on signaling activation. We have also worked to establish novel diagnostic strategies for follicular lymphoma. Emerging technologies allow broad profiling of the cancer genome for differential DNA methylation relative to benign cells. Bisulfite modified DNA from lymph nodes with either reactive hyperplasia or follicular lymphoma (FL) were analyzed using a commercial CpG/UpG genotyping assay. 259 differentially methylated targets (DMT) were identified in FL, distributed among 183 unique genes. Comparison of matched formalin-fixed-paraffin-embedded (FFPE) and frozen surgical pathology replicates demonstrated the complete preservation of the cancer methylome among differently archived tissue specimens. Analysis of the DMT profile is consistent with a pathologic process that affects predominantly non-lymphoid genes. This large-scale analysis of the lymphoma methylome identifies numerous DMT, provides novel insight into the epigenetic profile of a cancer tissue, and establishes the value of this approach to interrogate archived clinical cancer specimens.
Showing the most recent 10 out of 90 publications
