Hypermutation Status Research Articles

TCL1 Expression in B-Cell Tumors Is Dynamically Regulated by Maturation Stage, Proliferation, and the Follicular Microenvironment.

The kinase comodulator TCL1 is the primary initiating oncogene in T-cell prolymphocytic leukemia and can produce B-cell or T-cell chronic lymphocytic leukemia (CLL) following transgenic expression in mice. Given its strong expression in some non-neoplastic B-cell subsets, the role of TCL1 as an oncogene in human B-cell tumors is less clear. Using a recently developed TCL1 monoclonal antibody (clone 1–21), we examined the relationship between TCL1 expression and B-cell maturation stage in tumor tissue arrays, lymphoma cell lines, primary tumor samples and in vitro stimulation assays. Results were compared with immunohistochemical expression of a variety of maturation, activation and cell proliferation markers and with the somatic hypermutation status determined by VH transcript analysis (<2% sequence divergence in VH regarded as “pre-germinal center”). In non-neoplastic B-cells, TCL1 was strongly and uniformly expressed in naive B-cell subsets, but was variably expressed in subsets of germinal center B-cells, with complete absence of expression in immunoblasts and plasma cells. In germinal center B-cells, TCL1 was expressed more strongly in the quiescent centrocyte fraction than in the proliferating centroblasts. This pattern was replicated in human B-cell lymphoma lines and tumors with complete absence of TCL1 in myeloma cases (n = 35) and monocytoid B-cells in marginal zone lymphoma (n = 8) and dim or absent expression in the majority of the mutated/post-germinal center subset of B-CLL (10/15, 67%). In contrast, TCL1 showed strong but modulated expression in the majority of unmutated/pre-germinal center type of B-CLL (14/19, 74%), and mantle cell lymphoma (MCL, 44/57, 84%), and less frequently in follicular lymphoma (FL, 21/47, 45%). In B-CLL, TCL1 was overexpressed in non-proliferating cells within the pseudo-follicular proliferation centers but was markedly downregulated in the CD23+bright PCNA+ proliferating tumor cell component. Similarly in MCL, TCL1 was downregulated in the proliferative component but upregulated in tumor cells in follicular and mantle zone locations versus the diffuse areas. In FL, the highest levels of TCL1 expression were found in those cases that most strongly expressed the germinal center markers CD10 and bcl-6, but TCL1 staining was inversely correlated with expression of proliferation and activation markers, such as CD23. In 3 FL cases with multiple biopsies at different tissue sites, TCL1 was downregulated in tumor cells at extranodal sites as compared to those within lymph node follicles. Thus, the dynamic regulation of TCL1 in B-cells and derived tumors is likely due to changes in the balance between stimulatory microenvironmental influences within the lymphoid follicle and inhibitory signals during cell cycle progression/proliferation. In post-germinal center tumors, TCL1 expression is effectively silenced, and no longer exhibits this dynamic regulation pattern. These studies strongly suggest that TCL1 exerts its effects in promoting cell survival in quiescent B-cell subsets prior to and in the absence of an (antigenic) proliferative signal.

Heterogeneous somatic hypermutation status confounds the cell of origin in hairy cell leukemia

Hairy cell leukemia (HCL) is thought to arise from a post-germinal center (GC) B-cell, however the exact normal counterpart remains unclear. We performed V H gene analysis of 32 HCL cases, revealing somatically mutated V H genes (<98% homology) in 27 cases and unmutated V H genes in five cases, four of which displayed germline V H genes. Intraclonal heterogeneity was evident in the majority of eight mutated HCLs investigated, although at a lower level compared to GC-derived lymphomas. A novel finding of preferential V H3-30 gene usage was detected (19% of HCLs). Our data confounds the postulated post-GC origin in HCL considering (1) the finding of unmutated HCLs, generally correlating with a pre-GC origin, and (2) the presence of intraclonal variation in mutated HCLs. The latter suggests that the transformed B-cell was frozen when it still had an active mutation process, implying a closer relation to the GC than previously assumed. Furthermore, restricted V H3-30 usage indicates that antigen selection could be a promoting factor in HCL development.

Thymic function and immunoglobulin mutation genotype in B-cell chronic lymphocytic leukemia patients.

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by a profound dysregulation of the host's immune system at both the humoral and cellular level. We investigated to see if this dysregulation could be due partly to thymus dysfunction by quantifying the number of signal joint T-cell receptor excision circles (sjTRECs) in peripheral blood mononuclear cells of 30 untreated B-CLL patients at diagnosis and in age-matched healthy controls. sjTRECs were found decreased, normal and elevated in 19, 9 and 2 patients, respectively, in comparison to age-matched controls. We next speculated that sjTREC levels might be related to an accredited B-CLL prognostic marker represented by the somatic hypermutation (SHM) status of the variable heavy chain (V(H)) genes. Eight of 17 patients with SHMs had sjTREC levels in the range of or higher than normal donors, whereas only 3 of the 13 patients lacking SHMs had normal sjTREC levels. After a 5-year observation period in 16 patients for whom a clinical follow-up was available, only 2 of 10 patients with SHMs progressed vs. 5 of 6 patients without SHMs and 3 of 7 patients with normal or higher sjTREC levels progressed vs. 4 of 9 with low sjTREC levels. Our study demonstrates that sjTREC levels are decreased in >60% of B-CLL patients and suggests a potential role of thymus in the immune dysfunction of these patients.