Many issues and paradoxes need to be further investigated to understand the dynamic progression to overt follicular lymphoma. How does BCL2 overexpression combine to common follicular lymphoma-associated abnormalities to modify the dynamics of B cells in the germinal center? How does aberrant SHM target a cell with a light zone gene expression profile? What BCR and microenvironmental signals drive t(14;18)+ memory B-cell export from the germinal center and their subsequent re-entry? Answers to these questions will provide ways to specifically target the functional programs underlying follicular lymphoma progression, dissemination, and relapses.
Recent studies in mice have demonstrated that memory B cells are more diverse than previously considered , and challenged the common view that germinal centers are required for long-lived memory B-cell formation . Because memory/activated B cells, albeit of a particular CD5+ subset , are believed to be the normal counterpart of CLL cells, further understanding of germinal center-independent versus germinal center-dependent normal memory B-cell genesis should help to better understand their likely tumoral counterparts, namely U-CLL and M-CLL (reviewed in ).
BCR signaling plays a major role in CLL development and progression (reviewed in ). Circulating CLL cells of the U-CLL and M-CLL subsets have unusual glycosylation patterns of their sIgM receptors , decreased sIgM expression with some degree of functional anergy, all indicative of recent BCR engagement in vivo. Whether BCR activation in CLL is triggered by foreign antigen , self-antigen, or antigen-independent cis-interactions of sIgM  may be dependent on the U-CLL or M-CLL subsets and on the specific stereotypic Variable–Diversity–Joining rearrangements expressed [46,50]. Regardless, CLL physiopathology involves a dynamic cycle of BCR-induced proliferation in lymph node proliferation centers and recirculation in peripheral blood. Most interestingly, lymph node involvement is also seen in MBL cases (MBL in situ), most often with diffuse or interfollicular patterns and the presence of proliferation centers [51,52], suggesting the early role of local lymph node activation in CLL pathogenesis. Nevertheless, the signals triggering local BCR activation of MBL remain poorly defined and can only be inferred from CLL studies (Fig. 2 b). Recent gene expression profiling analyses of coupled CLL cells in blood, lymph node, and bone marrow have shown that the lymph node microenvironment induces BCR-signaling and pro-inflammatory gene expression signatures associated with more aggressive disease [53,54]. Transit through the lymph node induces higher expression of the miRNA miR-155, possibly through CD40L stimulation, which results in stronger BCR-induced signaling in CLL cells . The level of miR-155 expression gradually increased from normal B cells to MBL and CLL, suggesting that the assessment of circulating miRNAs may be used to predict which patients with MBL will go on to develop overt CLL . Circulating CLL cells can be staged based on their level of sIgM expression, with sIgM levels being positively correlated with CXCR4 expression and BCR-signaling responsiveness, and negatively correlated with expression of the proliferation marker Ki67 . These results favor a model of CLL physiopathology in which local activation of CLL cells in lymph node proliferation centers induces sIgM and CXCR4 downregulation and egress into the circulation, where sIgM and CXCR4 levels recover before iterative re-entry to lymph node (Fig. 2 B). In a mouse model of CLL (Eμ-Tcl1 mice), the follicular homing receptor CXCR5 controls CLL cell access to lymph node and spleen FDC-rich niches in primary follicles and germinal center light zone, in which bi-directional signaling is necessary for BCR-induced proliferation of CLL cells and lymphotoxin-mediated FDC activation and survival . Although CLL proliferation centers are distinct from germinal center structures, the mutating enzyme activation-induced cytidine deaminase (AID) is expressed in some locally activated CD86+ CXCR4low CLL cells (mostly M-CLL, although not strictly restricted to that subset) and may be responsible for intraclonal variation and DNA damage [59,60]. In fact, antigen-specific germinal center-independent memory B cells carry either IgM or switched isotypes, demonstrating that AID activity and class-switch recombination can proceed independently of germinal center formation [61,62].
Altogether, the data are consistent with a germinal center-independent memory B-cell chronic activation pathway driving premalignant CLL-like B-cell expansion in proliferation centers and progression from MBL to CLL. Many pending questions need to be addressed to better understand the dynamics of early CLL progression. Does CD5 expression poise naive or memory B cells towards an early germinal center-independent pathway upon activation? How does the status of the sIgM expressed by CLL cells (unmutated or mutated) affect premalignant cell dynamics? Germinal center-independent memory B-cell generation is independent of BCL6+ TFH cells but still requires CD4+ T cell help . Which T-cell subsets are involved in proliferation centers? How do genes recurrently targeted in CLL regulate the dynamic behaviors of CLL-like cells? Solving these issues will require innovative genetic and functional analyses in human samples and experimental models of early CLL.
The current effort to understand the early history of disease development in indolent malignancies points to a role of CPCs as disease reservoirs in relapse and transformation. It is therefore essential to investigate the pathophysiology of premalignant cell dynamics in order to identify targetable pathways that may hinder the capacity of these cells to evolve, disseminate or transform. As preleukemic HSC/progenitor cells bearing initiating mutations in leukemia-associated genes might represent another reservoir for disease relapse, new effective therapies are needed to selectively kill those CPC that are the real source of chronic leukemia or lymphomas.
WGS and WES of bulk samples have allowed to track the clonality of mutations in tumors through the quantification of variant allele frequency, but these approaches are rather limited in their ability to track rare subclonal SNV and determine whether they target independent clones. Thanks to considerable progress in next-generation sequencing and microfluidics it is now possible to sequence genomes from hundreds of single cells. Single-cell approaches have revealed clonal evolution in breast cancer primary tumors  and xenografts  with unprecedented resolution and likely start a new era in cancer biology. Single-cell genotyping also emerges as a simpler yet powerful approach to track clonal distribution of SNVs and CNAs (previously identified in WES and SNP-array analyses) during tumor progression [65,66]. It will be highly informative to study the genetics of premalignant B cells/HSCs at single-cell resolution in healthy persons and during disease progression. Such approaches should decipher the early genetic events driving lymphoma and leukemia development and characterize whether premalignant cells constitute a disease reservoir for relapse and transformation. Single-cell gene expression profiling, either by RNA sequencing or targeted qPCR, can also yield precious information on the functional heterogeneity of hematopoietic and immune cells [67–70]. It should now be feasible to compare the programs and signaling pathways in premalignant cells and identify potential therapeutic targets.
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