INTRODUCTION
Advances in high-throughput DNA analysis methods have permitted a more complete view of the complex genetic landscape of many tumor types, and revealed subclonal architectures in many cancers with important impact on prognosis (reviewed in [1]). These complex genetic traits likely evolve over many years within premalignant cells and remain undetected until full-blown disease is diagnosed. Screenings of blood DNA from large cohorts by whole exome sequencing (WES) have identified clonal hematopoiesis with recurrent age-related mutations in genes commonly associated with hematologic disorders (DNMT3A, TET2, ASXL1), their presence being associated with an increased risk of hematologic malignancies [2–4]. Because of their slow evolution, indolent B-cell malignancies provide unique opportunities to elucidate the premalignant history of disease evolution.
Here, we review the most recent literature describing premalignant cell dynamics in follicular lymphoma and chronic lymphocytic leukemia (CLL), two indolent B-cell malignancies characterized by a slow clinical course associated with multiple relapses, becoming increasingly resistant to therapy. Both diseases are preceded by a long asymptomatic preclinical phase during which premalignant B cells may (or not) accumulate additional genetic events and progress into overt tumors. We propose two distinct models involving perverted germinal center/memory B-cell responses that may explain the observed dynamics of premalignant cells in follicular lymphoma and CLL.
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PREMALIGNANT t(14;18)+ B CELLS AND FOLLICULAR LYMPHOMA DEVELOPMENT
Follicular lymphoma is a germinal center-derived B-cell malignancy in which the acquisition of the t(14;18) translocation – that lays the BCL2 gene under the transcriptional control of immunoglobulin heavy chain gene (IGH) regulatory regions – constitutes a critical early event in the natural history of follicular lymphoma. Yet, it is now well established that t(14;18) is also detectable at low frequency (1 per million lymphocytes) in up to 70% of healthy adults, indicating that t(14;18) is not enough to transform B lymphocytes into lymphomas. In rare cases, ‘healthy’ individuals carry unusually high frequencies of t(14;18)+ cells in the blood and those cells represent an expanding clonal population of atypical germinal center-derived memory B cells, mimicking genotypic and phenotypic features of follicular lymphoma cells. In particular, the so-called follicular lymphoma-like cells underwent class-switch recombination events while retaining a functional surface IgM (sIgM) allele that may contribute to malignant transformation. Whether some (and/or which) individuals carrying follicular lymphoma-like cells are prone to evolve into clinical follicular lymphoma and the time frame of progression remained so far largely circumstantial. Elevated levels of blood t(14;18) translocation were observed incidentally several years before follicular lymphoma diagnosis [5]. In a recent study, we compared t(14;18) frequency in the blood from healthy individuals who developed follicular lymphoma several years after sample archival to individuals who did not. We found significant higher levels of t(14;18) in prediagnostic samples and defined a t(14;18) frequency threshold above which there was a 23-fold higher risk of developing follicular lymphoma [6] (Fig. 1a). Thus, t(14;18) frequency provides a first marker to recognize committed follicular lymphoma precursors that will progress to follicular lymphoma. Clonal identity between premalignant t(14;18)+ B cells and follicular lymphoma was systematically found, suggesting that committed follicular lymphoma precursors can circulate at least 10 years before diagnosis. Direct evidence for such committed follicular lymphoma precursors was revealed in a case of donor-derived follicular lymphoma occurring concomitantly in both the donor and the recipient 9 years after hematopoietic stem cell (HSC) transplantation [7]. The same t(14;18) translocation was found in both donor and recipient's tumors, revealing a common origin for both follicular lymphomas. It was also found at high frequency in donor-lymphocyte infusions (DLIs) given to the recipient 7 years before follicular lymphoma diagnosis, demonstrating that commitment can occur many years before clinically manifest follicular lymphoma. Most importantly, in addition to t(14;18), deep sequencing of both follicular lymphoma tumors revealed the presence of at least 14 mutations shared between both tumors and the DLIs, suggesting that commitment to follicular lymphoma might require a complex set of alterations. Interestingly, analysis by ultra-deep sequencing of CD19−CD10−CD34+ cells from the DLIs, containing multipotent progenitors and HSCs, showed that a significant fraction of those cells already harbored three follicular lymphoma-associated mutations (TLN2, EP300, KLHL6) [8]. Thus, premalignant follicular lymphoma-initiating B cells might develop from more ancestral mutated HSCs with increased genetic instability and B-cell differentiation potential.
FIGURE 1: Follicular lymphoma (FL) and chronic lymphocytic leukemia (CLL) are preceded by premalignant B-cell clonal expansions that likely occur during a protracted preclinical phase of asymptomatic growth. BCR, B-cell receptor; tFL, transformed follicular lymphoma.
Recently, more comprehensive WES studies of paired diagnosis, relapsed, and transformed follicular lymphoma and thorough analyses of SNV have defined a more complete picture of the clonal patterns of follicular lymphoma evolution [9–11]. One major finding is that most transformed follicular lymphoma do not directly evolve from follicular lymphoma, but rather emerge from a common precursor clone (CPC) that is also ancestral to the dominant follicular lymphoma clones at diagnosis and relapse [10,11]. Also striking are the recurrent mutations of epigenetic regulators (e.g., MLL2, CREBBP, EZH2) that seem to drive early progression of follicular lymphoma and are likely to be present in the CPC. Altogether, the observed patterns of clonal evolution and early and late acquisition of mutations in key additional driver genes suggest a protracted yet very dynamic process leading from follicular lymphoma-like/CPC to overt follicular lymphoma/transformed follicular lymphoma.
PREMALIGNANT MONOCLONAL B-CELL LYMPHOCYTOSIS AND HEMATOPOIETIC STEM CELLS IN CLL
CLL is a low-grade B-cell malignancy characterized by the accumulation of mature clonal CD5+ CD19+ B cells in peripheral blood and lymphoid organs (lymph node and bone marrow). CLL cells have a gene expression profile resembling memory B cells, but can carry either unmutated (U-CLL) or mutated (M-CLL) B-cell receptor (BCR), pointing to a distinctive pre-germinal center or post-germinal center mature B-cell origin, respectively. Clonal accumulation of mature B cells in peripheral blood of healthy patients with cell counts less than 5 Ă— 109 B cells/L, a condition termed ‘monoclonal B-cell lymphocytosis’ (MBL), almost always precedes CLL [12,13], although most MBL cases never progress to overt CLL. Only high-count MBL is associated with a 1–2% per year risk of evolving into CLL requiring treatment (Fig. 1b) and can be considered as precursor CLL disease. Somatic copy number alterations characteristic of CLL, such as del(13q14) or trisomy 12, are also found in MBL with overall similar frequencies [13,14] (reviewed in [15]). WES has identified the most common mutations in CLL (affecting TP53, SF3B1, NOTCH1, ATM, MYD88, and BIRC3) [16–18]. Their likely order of appearance throughout pathogenesis has been inferred by analyzing clonal or subclonal distribution of mutations in longitudinal studies of CLL cases [19,20]. However, the most common mutations are only found in up to 15% of CLL cases on average, and have been found with a much lower incidence in CLL-like high-count MBL [14]. Thus, although high-count MBL already present some genomic alterations characteristic of CLL, the transformation into CLL likely follows a dynamic course involving the acquisition of additional genetic hits.
As for follicular lymphoma, development of clonally related donor-derived CLL or high-count MBL in both donor and recipient several years after allogeneic peripheral blood stem cell transplant also illustrates that committed precursor CLL cells may lie undetected in blood or in HSCs [21,22]. Actually, there is compelling evidence that HSCs may be the primary targets of initiating events leading to hematologic malignancies [23], including CLL. First, HSC from CLL patients have a bias for generating pro-B cells in culture systems and xenografts, and these cells produce monoclonal or oligoclonal B-cell progeny with many CLL/MBL features (phenotype, BCR repertoire) upon xenografting [24]. Second, circulating CD34+ early hematopoietic cells from CLL patients harbor CLL-associated mutations (BRAF, NOTCH1, EGR2, and SF3B1 among others), some of which can confer deregulated BCR signaling to mature B cells and poor prognosis [25â–ª].
HIJACKING GERMINAL CENTERS: t(14;18)+ CELL DYNAMICS IN FOLLICULAR LYMPHOMA PROGRESSION
Recent studies in mice have revealed that germinal centers are dynamic structures in which germinal center B cells shuttle back and forth between dark zone and light zone during BCR affinity maturation. Dark zone-based somatic hypermutation (SHM) and proliferation are followed by light zone-based antigen capture on follicular dendritic cells (FDCs) and cognate interactions with antigen-specific T follicular helper (TFH) cells [26]. Then, successful selection after TFH contact induces dark zone re-entry for another round of SHM [27,28]. In light of the numerous proliferative and survival pathways activated downstream of the BCR, it comes as no surprise that malignant B cells would co-opt this receptor to promote growth and survival (reviewed in [29]). There is a large body of evidence that follicular lymphoma progression is driven by a perverted B-cell immune response. First, although the BCR of follicular lymphoma cells is not stereotyped as for CLL cells, it is never lost despite extensive SHM. There is also evidence for self-antigen specificity in follicular lymphoma [30,31] and follicular lymphoma cells have an enhanced capacity to signal through their BCR compared to nonmalignant follicular B cells [32]. Second, bidirectional and dynamic interactions with the stromal and T-cell microenvironment contribute to follicular lymphoma pathogenesis through enhancing survival and proliferation of follicular lymphoma cells (reviewed in [33]). The frequent introduction of N-linked glycosylation sites created by SHM in the immunoglobulin variable gene region of follicular lymphoma BCRs might favor BCR activation through lectin binding from the microenvironment [34]. Most follicular lymphoma-like and follicular lymphoma cells express BCR of IgM isotype (70%), and thus may be more prone to re-enter into germinal center as has been observed for mouse IgM memory B cells [35]. The fortuitous findings of germinal center structures filled with BCL2+CD10+ follicular lymphoma-like cells adjacent to normal GCs in hyperplastic lymph node support this capability of pre-follicular lymphoma cells to be mobilized into open GCs. This preclinical entity, named ‘follicular lymphoma in situ’ (FLIS), is believed to represent an intermediate stage in follicular lymphoma pathogenesis [36].
These observations led us to propose a model of early follicular lymphoma pathogenesis in which chronic antigen stimulation of t(14;18)+ B cells would promote iterative germinal center re-entries, gradually increasing the risk of accumulating somatic mutations throughout a lifetime of immunological challenges [20]. We have recently generated an original sporadic BCL2tracer mouse model, mimicking the rare occurrence of t(14;18) in humans allowing to track BCL2-expressing clones during chronic activation. We have shown that cycles of germinal center re-entries of BCL2+ germinal center/post-germinal center memory cells over an extended period of time lead to the accumulation of SHM and formation of FLIS-like structures [37▪]. Moreover, we measured IGH sequence diversity in t(14;18)+ B cells and conventional memory B cell clones in paired human blood and lymphoid tissues from organ donors, and showed that t(14;18)+ B-cell clones widely disseminated in lymph node, spleen, and even bone marrow had increased intraclonal variation patterns [37▪], compatible with iterative rounds of germinal center co-opting, in which t(14;18)+ cells accumulate SHM in their IGH genes (and possibly off-target oncogenic mutations). Germinal center B cells are normally strictly confined to the germinal center microenvironment, but long-lived memory B cells and plasma cells circulate and home to other secondary lymphoid organs and the bone marrow, respectively. Germinal center confinement is mediated by inhibitory signaling via Gα13-coupled receptors like the sphingosine-1-phosphate receptor S1PR2 and the orphan receptor P2RY8, a pathway that is frequently targeted by genetic alterations in germinal center-derived lymphoma, explaining why most germinal center-derived lymphomas (and possibly premalignant follicular lymphoma cells) widely disseminate and escape germinal center confinement [38▪▪]. In addition, t(14;18)+ B cells home preferentially to the germinal centers from nonmalignant reactive hyperplasia and mostly within the nonproliferating light zone subset [39], suggesting their likely participation in immune reactions. It is worth noting here that most germinal center B-cell lymphomas, including follicular lymphoma, seem to be more closely related – based on gene expression profiling – to light zone B cells than dark zone B cells [40]. Interestingly, physical access to the dark zone is not strictly required for germinal center B-cell proliferation and SHM, indicating that the light zone environment can also provide the necessary factors for affinity maturation [41]. A model of early follicular lymphoma development that involves B-cell dynamic subversion that culminate in FLIS and follicular lymphoma is proposed (Fig. 2 a).
FIGURE 2: (a) Model of memory B-cell dynamics of BCL2/IGH carrying cells in early follicular lymphoma (FL) pathogenesis. Upon antigenic stimulation, naive t(14;18)+ cells enter the germinal center and undergo proliferation, somatic hypermutation (SHM) [and for some cells class-switch recombination (CSR)], leading to affinity maturation of their B-cell receptor (BCR). In the light zone, enforced BCL2 expression rescues t(14;18)+ cells of low BCR affinity and allows cells to differentiate into switched and mostly IgM memory B cells. Upon recall, regularly occurring in human life, t(14;18)+ IgM memory B cells are preferentially reactivated to enter iterative cycles of germinal center reaction or hijack reactive germinal centers, before acquiring a germinal center-associated developmental arrest and constitutive activation-induced cytidine deaminase (AID) ‘mutator’ activity. Such dynamics will gradually induce clonal expansion, early systemic dissemination, and progression in t(14;18)+ precursor cells that culminate in ‘follicular lymphoma in situ’ (FLIS) and FL. (b) Model of chronic BCR stimulation governing evolution from monoclonal B-cell lymphocytosis (MBL) to chronic lymphocytic leukemia (CLL) pathogenesis. Primary genetic alterations leading to CLL may be acquired at an early self-renewing CLL-hematopoietic stem cell (HSC) stage and prime cells toward a biased B-cell differentiation, leading to naive CD5+ B cells with CLL-like mutations. Depending on the nature of the BCR antigenic stimuli and stereotypy, CD5+ B cells will be driven either into a classical germinal center-dependent reaction in which cells exit mostly as IgM+ memory B-cell phenotype with mutated BCR. Alternatively, cells might enter a germinal center-independent pathway and exit as activated B cells without immunoglobulin variable gene (IgV) mutations. Rounds of proliferation centers re-entry and ongoing antigenic stimulation through the BCR might contribute to clonal expansion of CLL-like cells in blood and tissue. Local AID expression in proliferation centers would favor progressive genomic instability in MBL clones, while sparing BCR in unmutated cases, ultimately leading to CLL. Unmutated CLL-like cells, having a higher propensity to respond to BCR stimulation, will evolve more rapidly to a progressive UM-CLL, whereas mutated CLL-like cells might operate more slowly to evolve into MBL. IGH, immunoglobulin heavy chain gene; VDJ, Variable–Diversity–Joining.
FIGURE 2: (a) Model of memory B-cell dynamics of BCL2/IGH carrying cells in early follicular lymphoma (FL) pathogenesis. Upon antigenic stimulation, naive t(14;18)+ cells enter the germinal center and undergo proliferation, somatic hypermutation (SHM) [and for some cells class-switch recombination (CSR)], leading to affinity maturation of their B-cell receptor (BCR). In the light zone, enforced BCL2 expression rescues t(14;18)+ cells of low BCR affinity and allows cells to differentiate into switched and mostly IgM memory B cells. Upon recall, regularly occurring in human life, t(14;18)+ IgM memory B cells are preferentially reactivated to enter iterative cycles of germinal center reaction or hijack reactive germinal centers, before acquiring a germinal center-associated developmental arrest and constitutive activation-induced cytidine deaminase (AID) ‘mutator’ activity. Such dynamics will gradually induce clonal expansion, early systemic dissemination, and progression in t(14;18)+ precursor cells that culminate in ‘follicular lymphoma in situ’ (FLIS) and FL. (b) Model of chronic BCR stimulation governing evolution from monoclonal B-cell lymphocytosis (MBL) to chronic lymphocytic leukemia (CLL) pathogenesis. Primary genetic alterations leading to CLL may be acquired at an early self-renewing CLL-hematopoietic stem cell (HSC) stage and prime cells toward a biased B-cell differentiation, leading to naive CD5+ B cells with CLL-like mutations. Depending on the nature of the BCR antigenic stimuli and stereotypy, CD5+ B cells will be driven either into a classical germinal center-dependent reaction in which cells exit mostly as IgM+ memory B-cell phenotype with mutated BCR. Alternatively, cells might enter a germinal center-independent pathway and exit as activated B cells without immunoglobulin variable gene (IgV) mutations. Rounds of proliferation centers re-entry and ongoing antigenic stimulation through the BCR might contribute to clonal expansion of CLL-like cells in blood and tissue. Local AID expression in proliferation centers would favor progressive genomic instability in MBL clones, while sparing BCR in unmutated cases, ultimately leading to CLL. Unmutated CLL-like cells, having a higher propensity to respond to BCR stimulation, will evolve more rapidly to a progressive UM-CLL, whereas mutated CLL-like cells might operate more slowly to evolve into MBL. IGH, immunoglobulin heavy chain gene; VDJ, Variable–Diversity–Joining.
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.
GERMINAL CENTER-INDEPENDENT MEMORY B-CELL DYNAMICS IN CHRONIC LYMPHOCYTIC LEUKEMIA PROGRESSION
Recent studies in mice have demonstrated that memory B cells are more diverse than previously considered [42], and challenged the common view that germinal centers are required for long-lived memory B-cell formation [43]. Because memory/activated B cells, albeit of a particular CD5+ subset [44], 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 [45]).
BCR signaling plays a major role in CLL development and progression (reviewed in [46]). Circulating CLL cells of the U-CLL and M-CLL subsets have unusual glycosylation patterns of their sIgM receptors [47], 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 [48], self-antigen, or antigen-independent cis-interactions of sIgM [49] 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 [55]. 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 [56]. 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 [57]. 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 [58]. 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 [61]. 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.
CONCLUSION AND FUTURE DIRECTIONS
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 [63] and xenografts [64] 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.
Acknowledgements
None.
Financial support and sponsorship
This work was supported by the Institut National du Cancer (INCa, France), the Association pour la Recherche sur le Cancer (ARC, France), the Fondation de France, a MedImmune (Gaithersburg, Maryland, USA) Strategic Collaboration to Fund and Conduct Medical Science Research program, and institutional grants from the Institut National de la Santé et de la Recherche Médicale (INSERM) and the Centre National de la Recherche Scientifique (CNRS). P.M. is supported by a fellowship from ARC.
Conflicts of interest
There are no conflicts of interest.
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Papers of particular interest, published within the annual period of review, have been highlighted as:
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