B cell receptor signaling proteins as biomarkers for progression of CLL requiring first-line therapy

Abstract

The molecular landscape of chronic lymphocytic leukemia (CLL) has been extensively characterized, and various potent prognostic biomarkers were discovered. The genetic composition of the B-cell receptor (BCR) immunoglobulin (IG) was shown to be especially powerful for discerning indolent from aggressive disease at diagnosis. Classification based on the IG heavy chain variable gene (IGHV) somatic hypermutation status is routinely applied. Additionally, BCR IGH stereotypy has been implicated to improve risk stratification, through characterization of subsets with consistent clinical profiles. Despite these advances, it remains challenging to predict when CLL progresses to requiring first-line therapy, thus emphasizing the need for further refinement of prognostic indicators. Signaling pathways downstream of the BCR are essential in CLL pathogenesis, and dysregulated components within these pathways impact disease progression. Considering not only genomics but the entirety of factors shaping BCR signaling activity, this review offers insights in the disease for better prognostic assessment of CLL.

Keywords:

• Chronic lymphocytic leukemia
• B cell receptor
• prognosis
• signaling pathways
• time-to-first-treatment

Introduction

Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disorder characterized by progressive accumulation of mature, monoclonal CD5⁺ B cells. Intervention at early-stage disease does not improve overall survival, hence treatment is only initiated after onset of disease-related symptoms or signs of rapid progression [1]. The time-to-first-treatment (TTFT), a term employed to describe the time interval from diagnosis to start of first-line therapy, may vary between a few months to multiple years from diagnosis, illustrating the clinical heterogeneity of CLL. Significant efforts have been made to discriminate indolent from aggressive CLL at diagnosis. Traditional clinical prognostic staging systems like the Rai and Binet classifications and lymphocyte doubling time are rough estimates for risk stratification, which have been later refined by the addition of advanced molecular diagnostics determining cytogenetics (such as deletions of 17p, 11q and 13q or trisomy 12), immunogenetics (immunoglobulin heavy chain variable gene (IGHV) mutational status), and oncogenetic lesions (TP53, NOTCH1, and others) [1]. Despite the inclusion of some of these prognostic factors, it still remains challenging to predict TTFT at early disease stages of CLL. Moreover, molecular biomarkers are frequently characterized only prior to treatment, rather than at the time of diagnosis.

Furthermore, genetic variants do not fully explain clinical heterogeneity, as genomic abnormalities are just one of the many hallmarks of cancer that establish aberrant signaling pathways to achieve tumor survival. Rather, clinical heterogeneity is shaped by the combination of epigenetic, genetic, and micro-environmental cues, which intersect at the intracellular signaling level. Therefore, measuring the activity of these dysregulated pathways may more accurately capture the pathogenic potential of each instance of CLL.

Although various proliferative signals are present in the leukemic micro-environment, B-cell receptor (BCR) signaling takes center stage [2,3]. This is emphasized by the remarkable clinical efficacy of BCR-associated kinase inhibitors that target and inhibit BCR signaling in the pathophysiology of CLL [4]. In this review, we will discuss how proteins involved in BCR signaling are associated with TTFT and may be used for prognostication of CLL.

Antigen receptor gene recombination and antigen response in healthy B cells

Before exploring BCR signaling proteins as biomarker in CLL, a brief recap of normal BCR formation and the consequences of antigen recognition is needed. Gene recombination of the immunoglobulin (IG) genes by B cells lays the foundation to an enormous antigen-specific BCR repertoire. Stochastic assembly of genes occurs early on in B cell development within the bone marrow, starting with rearrangement of the variable (V), diversity (D), and joining (J) genes of the IG heavy (IGH) chain locus. Here, the pro-B cell recombines one V, D, and J gene out of a range of available genes and ligates them together. Correct VDJ gene utilization and proper ligation yields the formation of a functional IGH chain. When combined with the surrogate light chain, this pre-BCR provides signals for survival and further maturation of the B cell. Following this positive selection procedure, the VJ-recombination of the IG kappa or lambda (IGK/IGL) light chain locus occurs. During V(D)J recombination, additional variety in BCR composition is induced by enzymatic editing of the junctional regions, referred to as complementarity determining region 3 (CDR3) [5]. Next, the IGH and IGK/IGL chains are combined and expressed on the membrane surface in a micro-cluster containing CD79α/β. This BCR complex is tested for antigen binding in the bone marrow, where inappropriately strong binding leads to clonal deletion or receptor editing to avoid self-reactivity. Survivors of this negative selection step, now mature B cells, are released into the peripheral circulation, where they recirculate in secondary lymphoid tissues, until they encounter their cognate antigen.

When a mature B cell recognizes and binds its cognate antigen, a sequential phosphorylation pattern of specific tyrosine kinases is triggered. First, Lyn phosphorylates the cytoplasmic tails of CD79α/β, which subsequently recruit Syk. Syk is key in BCR signaling and is activated near the membrane by phosphorylation. Syk in turn activates BTK, an interaction optimized by BLNK, and this complex then phosphorylates PLCγ2. The activated PLCγ2 isoform induces the second messengers PKC and Ca²⁺ for activation of ERK, JNK, p38, NF-κβ, and NFAT. Syk simultaneously activates PI3K, which initiates the AKT pathway, this process is contingent upon CD19 phosphorylation by Lyn [6,7]. Collectively, these actions will establish an intracellular survival program for the B cell following an appropriate antigen encounter. Moreover, in certain instances, as determined by the nature of the antigen and contextual cues, somatic hypermutation (SHM) commences in the germinal center of the lymph node follicle. This process is initiated for additional diversification of the BCR repertoire. During SHM, point mutations are introduced in the V genes of the IGH and IGK/IGL chains to optimize antigen binding [5].

Finally, immunogenic tolerance mechanisms come into play if a BCR engages with an epitope without proper co-stimulation, or if this engagement is not of the required strength. In such instances, the B cell undergoes apoptosis, a form of programmed cell death, or enters a state of anergy, rendering the cell non-responsive [8].

Immunogenetic profiling for risk stratification of CLL

The first piece of evidence on the importance of BCR signaling in CLL stems from immunogenetic studies, in which the distinctive features of the clonotypic BCR provided significant insights into the leukemogenesis and prognosis of CLL. Two hallmark studies in the late nineties documented that the SHM imprint on the IGHV gene, which allows classification of CLL as either IGHV unmutated (U-CLL; ≥98% germline homology) or IGHV mutated (M-CLL; <98% germline homology), is strongly associated with TTFT [9,10]. On average, U-CLL patients require treatment more often and substantially earlier than M-CLL patients. This could be related to an increased incidence of recurrent gene mutations with adverse outcome in U-CLL [11,12], or alternatively, attributed to differential BCR signaling capacity. These hypotheses could be interlinked, as the SHM status might just mirror distinct ontogenies of the two subsets. For example, the IGHV SHM load could indirectly reflect the maturation stage of the B cell before leukemic transformation, differentiating early- from late-stage memory B cell precursors [13]. On the other hand, it could indicate the type of B cell response, where germinal center reactions are different from extra-follicular reactions [14]. These discrepancies result in unique epigenetic, transcriptomic, and metabolic phenotypes of which those fitting U-CLL have a higher predisposed potency for clonal expansion and evolution, ultimately leading to an increased risk of disease progression.

Strikingly, CLL-associated BCR rearrangements are characterized by the use of restricted V(D)J gene usage. IGHV gene repertoire analysis of CLL cases revealed predominance of specific IGHV genes (IGHV1-69, IGHV3-7/3-23, and IGHV4-34 being the most frequent). Moreover, unrelated CLL patients often carry quasi-identical BCR rearrangements, which is referred to as BCR stereotypy [15]. The CLL cells of patients within these ‘stereotyped subsets’ share the same immunogenetic features, like IGHV/IGHD/IGHJ gene usage and HCDR3 sequence [15–18]. Major subsets even display remarkable sequence restrictions limited to just a few critical amino acid residues in the HCDR3 [15,17]. Currently, one-third of patients may be grouped into subsets based on HCDR3 sequence motifs, of which major subsets #1, #2, #4, and #8 are collectively the largest fraction (6–7%) in CLL [15]. Recent reports suggest that this frequency may rise with larger studied cohort sizes, effectively implying that the vast majority of CLL can be grouped into stereotypic clusters (Agathangelidis and Stamatopoulos et al., unpublished). Interestingly, patients within the same subset exhibit consistent clinico-biological profiles. For instance, patients from subset #2 often have a more aggressive disease course, independent of IGHV SHM status. This highlights BCR stereotypy as an additional prognostic marker for stratification of an otherwise heterogeneous disease [16, 18,19].

So far, BCR stereotyped subsets are defined on the basis of their IGH chain rearrangement. However, stereotyped IGH-chain BCR rearrangements are frequently paired to specific IGK/IGL light chains. For example, subset #2 BCRs are always a combination of an IGHV3-21 heavy-chain and an IGLV3-21 lambda light chain, featuring the recently described and clinically relevant R110 SHM [20,21]. Although these observations suggest that, in the context of CLL, the clonotypic IGK/IGL rearrangement may also be important, the prognostic impact of the CLL IGK/IGL chain repertoire has been relatively understudied so far.

Antigen-dependent signaling in CLL

The aforementioned asymmetry of IGHV gene usage, combined with the observed HCDR3 restriction, may be indicative of an antigenic drive shaping the clonal BCR repertoire in CLL. Indeed, transcriptional, epigenetic, and immunophenotypic studies revealed that CLL cells resemble antigen-experienced B cells [22]. Additional investigation identified distinct antigens that are recognized by CLL clones. M-CLL cells have endured antigenic pressure, by which SHM generates high-affinity binding and more restricted reactivity. However, such affinity matured cells would encounter their antigens less frequently due to the specificity of their BCR. Hence, the sporadic encounters, combined with higher affinity binding, often lead to apoptosis or anergy. On the contrary, U-CLL is mostly characterized by polyreactive BCRs. It is plausible that increased polyreactivity raises the likelihood of very frequent interaction with an antigen. This persistent exposure, coupled with lower affinity binding, may result in increased proliferation and therefore shorter TTFT, shorter response to chemo-immunotherapy, finally resulting in shorter survival of patients with U-CLL [23]. However, an alternative theory has been proposed to explain the divergent disease courses observed in M-CLL and U-CLL. According to this hypothesis, M-CLL cells undergo receptor editing to mitigate auto-reactivity, ultimately leading to a more stabilized disease state. This suggestion is grounded in research restoring the BCRs of M-CLL cells to germline sequences, which re-established polyreactivity [22]. That said, both concepts attribute the primary difference in prognosis between the two subsets to BCR promiscuity. Notably, polyreactivity of the BCR is independently associated with aggressive disease [24].

Further investigation into the disparities of BCR signaling between M-CLL and U-CLL concerned in vitro studies, where IgM cross-linking by antibodies was employed as surrogate for in vivo BCR ligation. The cellular response to IgM cross-linking is diverse, eliciting no response at all in ≈30% of CLL cases, while inducing apoptosis or proliferation in the remainder. Antigen-induced anergy is observed for both subsets, but it is more profound in M-CLL, likely related to the BCR features of this subset. This also explains why the responsive proportion of CLL is enriched for U-CLL cases [25,26].

Expression levels of BCR complex proteins

The inability to signal through the BCR is mostly established by low surface IgM expression [27], a feature related to chronic antigen engagement [28]. Increased surface IgM expression is correlated with worse disease outcome [29–31], although this may be dependent on a generally higher expression of IgM in U-CLL [3,27,32]. Evidence also exists for defective isoforms and assembly of CD79β, but the actual functional relevance of that has been controversial [33]. It is also important to note that failure to respond is not solely determined by IgM expression, as similarly expressing IgM cases were found to respond differently [27, 34]. Furthermore, CLL anergy is characterized by constitutive phosphorylation of BCR signaling mediators, transactivation of NFAT, retained IgD surface expression, elevated basal Ca²⁺ levels and increased negative feedback from the CD5/SHP-1 axis [32,35]. Importantly, non-responsiveness to IgM ligation is reversible in vitro by additional stimuli [36,37], a mechanism likely to occur within the tumor micro-environment, or can revert back spontaneously [27], possibly due to the termination of antigen engagement.

CLL cells with competent BCR signaling are more efficient at phosphorylation of the signalosome, as well as Ca²⁺ mobilization, following anti-IgM stimulation [26, 32, 34, 38]. This has pathophysiological relevance, given that the quantitative differences in BCR activity succeeding IgM modulation are predictive of TTFT, as is the case for the phosphoprofiles of tyrosine kinases [26, 32, 34, 39] and Ca²⁺ influx [31, 40] (Figure 1; Table 1). Responsiveness is attributed to the elevated levels of membrane and proximal signaling molecules before antigen-encounter, which ensures that the abnormal B cell is closer to reaching the response threshold [32]. As mentioned earlier, this is dominantly determined by IgM levels, but other receptors as part of the micro-cluster may also shape responsiveness (Figure 1; Table 1). One such molecule is CD20, higher levels of which predict favorable prognosis [41], although this may seem paradoxical, as reduced CD20 expression impairs kinase phosphorylation after BCR engagement in CLL [61]. As of now, the precise role of CD20 in CLL thus remains elusive.

Figure 1. Factors affecting BCR responsiveness in CLL that are associated with TTFT. BCR responsiveness is a gradient, of which the more extreme scenarios are depicted in the left and right panels. BCR non-responsive CLL cells are frequently M-CLL, have restricted antigen recognition and are positive for CD20 (left). BCR responsive CLL cells are typically U-CLL that are polyreactive, have high expression of adaptor proteins, and show elevated basal levels of primary BCR signaling components and other factors enhancing BCR signaling (right). These differences ultimately lead to increased tyrosine kinase phosphorylation and elevated Ca²⁺ mobilization following IgM stimulation in the responsive CLL cells, whereas non-responsive cases do not exhibit such changes. Created with BioRender.com. BCR: B cell receptor; CLL: chronic lymphocytic leukemia; IGHV: immunoglobulin heavy chain variable gene; M-CLL: IGHV-mutated CLL; TTFT: time-to-first-treatment; U-CLL: IGHV-unmutated CLL.

Table 1. Proteins affecting BCR signaling activity that are associated with TTFT in CLL.

Role in CLL BCR signaling	Measured	Univariate/multivariable analysis	Association with IGHV SHM statusa	Ref.	Impact on TTFTb
BCR complex	SHM load of ≥98% in IGHV gene	Univariate	N.a.	[9,10]	↑U-CLL
BCR complex	Subset #1, #2, #4 and #8	Univariate within M-CLL and U-CLL	Additive	[18,19]	↑subset #2
BCR complex	IgM MFI	Multivariable	Independent	[29–31]	↑
BCR microcluster	CD20 MFI	Univariate	Associated	[41]	↓
BCR synapse formation	CD38 MFI	Univariate/multivariable without IGHV SHM status	Not performed	[42]	↑
Ligand of BCR	Reactivity to ≥5 epitope mimics	Multivariable	Independent	[24]	↑
Primary BCR signaling cascade	Lyn mRNA	Univariate	Not associated	[43]	↑
Primary BCR signaling cascade	Syk phosphorylation following H2O2 exposure	Univariate	Not associated	[44]	↑
Primary BCR signaling cascade	Phosphorylation of Syk or ERK following IgM cross-linking	Multivariable	Dependent	[26, 39]	↑
Primary BCR signaling cascade	Phosphoprofile containing Syk, PLCγ2, ERK, BTK, and BLNK following IgM cross-linking	Univariate	Associated	[34]	↑
Primary BCR signaling cascade	NFAT2 DNA-binding activity following IgM cross-linking	Univariate	Not performed	[32]	↑
Primary BCR signaling cascade	CARD11 mRNA	Univariate	Not associated	[45]	↑
Primary BCR signaling cascade	Ca^2 influx following anti-IgM cross-linking	Univariate within M-CLL and U-CLL	Additive	[31]	↑
Calcium homeostasis	MZB1 mRNA	Multivariable	Dependent	[46]	↑
Signal amplification	ZAP-70 MFI	Multivariable/univariate within M-CLL and U-CLL	Independent/additive	[47]	↑
Signal amplification	SLP76 protein	Univariate	Associated	[48]	↑
Signal amplification	TCL1 protein	Multivariable	Independent	[49]	↑
Signal amplification	THEMIS2 protein	Multivariable	Independent	[50]	↑
Signal amplification	GAB1 mRNA	Univariate	Not performed	[51]	↑
Negative feedback	FcγRIIb MFI	Univariate	Not associated	[52]	↑
Negative feedback	CD5 MFI	Multivariable	Independent	[53]	↓
Negative feedback	LAIR-1 MFI	Multivariable	Independent	[54]	↓
Inhibitory	SLAMF1/7 MFI	Multivariable	Independent	[55]	↓
Inhibitory	ENDOG/PTEN mRNA	Univariate within M-CLL	Additive for M-CLL	[56]	↑ ENDOG ↓ PTEN
Inhibitory	FCMR MFI	Univariate within M-CLL and U-CLL	Additive for M-CLL	[57]	↓
Integration of signal	Transcriptome signature	Multivariable /univariate within M-CLL and U-CLL	Independent/additive	[58–60]	↑molecular signature of BCR signals

BCR: B cell receptor; CLL: chronic lymphocytic leukemia; IGHV: immunoglobulin heavy chain V gene; SHM: somatic hypermutation; MFI: median/mean fluorescent intensity; M-CLL: IGHV-mutated CLL; TTFT: time-to-first-treatment; U-CLL: IGHV-unmutated CLL.

^a Additive: biomarker shows significance after univariate analysis within the M-CLL or U-CLL subgroup. Associated: survival analysis did not include IGHV SHM status, but the biomarker was significantly different between M-CLL and U-CLL based on separate statistical analysis. Dependent: biomarker was assessed in multivariable analysis using the covariate IGHV SHM status and was not significant. Independent: biomarker was assessed in multivariable analysis using the covariate IGHV SHM status and remained significant.

^b ↑: high levels or presence of biomarker are associated to shorter TTFT. ↓: low levels or absence of biomarker are associated to shorter TTFT.

Setting the threshold for BCR signaling

Responsiveness to BCR stimulation may be further enhanced by proteins that amplify initially weak signals (Figure 1; Table 1). ZAP-70 has been implicated to modulate BCR signaling strength in CLL. Whether responsiveness is directly related to ZAP-70 has been difficult to unveil, as it is highly correlated to U-CLL and other co-factors, but focusing on the proficiency of IgM signaling in ZAP-70 positive M-CLL cases argued for an additional effect of ZAP-70 [62]. Syk and ZAP-70 are paralogs and expected to have redundant roles [63], but it was shown that ZAP-70 proves to be beneficial, even in high-expressing Syk cases [64]. In fact, ZAP-70 might stabilize phosphorylated Syk in a kinase-independent manner to prolong functioning [65,66]. The functional significance of ZAP-70 in BCR-mediated responses could explain why ZAP-70 expression is associated with TTFT of CLL patients independent of IGHV mutational status [47]. Strikingly, recent work contradicts this statement, demonstrating that ZAP-70 only increases tonic BCR signaling in the U-CLL subset, and this affect is not observed after BCR stimulation [67]. However, opposed to the previously mentioned works investigating ZAP-70, these authors used immobilized IgM (IgM-coated on beads), which is known to elicit stronger responses than soluble IgM [68]. This stimulus might be robust enough on itself and not require the signal amplification that ZAP-70 provides, thus explaining that inhibition of ZAP-70 during IgM stimulation did not alter responsiveness.

Like ZAP-70, SLP76 has the ability to augment BCR signaling as an adaptor protein. SLP76 binds Syk and BTK, an analogous activity to BLNK. Notably, SLP76 is associated to shorter TTFT [48].

The transmembrane glycoprotein CD38 is an important biomarker for aggressive disease in CLL, and has also been implicated in BCR signaling [42]. CD38 may be necessary for assembly of CD19 and other co-receptors in IgM synapses [69,70]. So far, discerning the actual influence of CD38 on responsiveness has been challenging, given that its effects are entangled with U-CLL classification [27]. To resolve this issue, a similar methodology as the one described by Chen et al. for ZAP-70 could be applied, in which M-CLL cases are transfected with CD38 expression.

The oncoprotein TCL1 has also been associated with TTFT in CLL. This association may stem from the recruitment of TCL1 to the BCR complex, where it guides the degree of AKT phosphorylation after IgM cross-linking [49]. However, it is important to note that TCL1 participates in various pathways, suggesting that its role in BCR regulation may not be the sole factor at play [71]. Nevertheless, it is noteworthy that the precision of predicting BCR responsiveness was remarkably high based on TCL1 kinetics following BCR aggregation [49].

Recently performed research by our group identified THEMIS2 as independent biomarker of TTFT in an untreated CLL cohort [50]. THEMIS2 is an adaptor protein that exaggerates BCR signaling by binding both Lyn and PLCγ2 to stabilize this interaction, a mechanism employed to increase sensitivity to low avidity or abundant antigens [72].

Finally, multiple additional proteins that act in the downstream BCR signaling cascade to fine-tune the threshold are associated with TTFT in CLL, such as GAB1, MZB1, and CARD11 [45,46, 51]. These three proteins operate in different downstream effector branches of the BCR signaling cascade. GAB1 is an adaptor molecule in the PI3K pathway, MZB1 controls Ca²⁺ homeostasis to regulate NFAT1 translocation and CARD11 is involved in NF-κβ signaling.

The composite effect of all these direct and indirect signaling mediators sets the threshold needed to be reached in order to elicit a cellular response. When this threshold is exceeded during BCR triggering, it leads to prolonged MEK/ERK, PI3K/AKT and to a lesser extent NF-κβ activation [73,74]. This response is incomplete when compared to healthy B cells, as no JNK or p38 pathways are set in motion [26, 74]. Exactly how strong the response is will guide the balance of pro- and anti-apoptotic proteins, with the main regulators of cell death being MCL-1, BCL2, and BIM in CLL [73–75].

Antigen-independent signaling in CLL

Apart from disruptions in antigen-dependent signaling, CLL might also exhibit aberrant antigen-independent signaling. Several studies have identified the existence of constitutively active components of the BCR signaling pathway in unstimulated CLL cells. Either increased basal levels or increased phosphorylation have been observed for Lyn [25, 75–77], Syk [25, 75, 78], BTK [79,80], and PLCγ2 [38, 78], as well as other BCR-signaling proteins [78], when compared to healthy B cells. Continuous propagation of signals through the BCR could theoretically be related to genetic aberrations in the primary signaling cascade, but the presence of such mutations has been excluded [77, 81].

Reconstitution of BCRs from CLL cases in a cell line model system revealed that CLL cells exhibit overactive BCR signaling in the absence of known antigens [82]. Further structural analysis detected homotypic interactions between neighboring BCRs at the cell membrane in CLL, resulting in self-recognition-triggering Ca²⁺ mobilization [83] (Figure 2(A)). To illustrate this phenomenon, we will use the CLL subset with the aforementioned IGLV3-21^R110 light chain as example. This subset is characterized by a single G to C nucleotide substitution at the cross-section between IGLJ3 (or IGLJ1) and the constant region in the lambda light chain, enabling the newly created R110 to bind D50 on a neighboring BCR. Furthermore, the presence of a K16 residue, derived from the IGLV3-21*01 or IGLV3-21*04 gene, is imperative for efficient autonomous signaling of the BCR by facilitating binding with the D52 residue [21]. While the contention may arise that homotypic BCR interactions are in fact recognizing an antigen, we interpret it as a distinct physiological process that differs from the conventional concept of antigen-dependent signaling. The term ‘antigen-independent’ is therefore used as collective descriptor for both tonic and homotypic BCR signaling in this respect. Of interest, IgM isotypes were identified as most potent in relying tonic signals, explaining why most CLL clones do not undergo class-switch recombination, even ones that experienced SHM. That said, class-switching from IgM to IgG is mandatory for subset #4 to be able to self-recognize, as the IgG isotype contains a K214 residue not conserved in IgM [83].

Figure 2. Antigen-independent, elevated basal BCR signaling in CLL and proteins impacting TTFT. Autonomous BCR signaling by homotypic interactions between cell-surface BCRs and tonic BCR signaling by spontaneous phosphorylation both contribute to survival of CLL cells (A). The dynamics of antigen-dependent and -independent signaling in CLL are reliant on the physiological location of the cell. Antigen-dependent signaling occurs more frequently in the lymph nodes resulting in proliferation, while antigen-independent signaling maintains CLL cells within the peripheral blood circulation till the cells re-enter the lymph nodes. The strength of the homotypic interaction determines the capacity of the CLL cell to survive within the blood (B). (B) Slightly modified from Haselager et al. [84]. Created with BioRender.com. BCR: B cell receptor; CLL: chronic lymphocytic leukemia; TTFT: time-to-first-treatment.

The relative contributions of antigen-dependent and -independent signaling are postulated to be different between CLL clones in a time- and strength-dependent manner, which may explain the substantial variability in the dynamics of many kinases downstream of the BCR across CLL cohorts [44, 85] (Figure 2(B)). Investigation of cellular kinetics demonstrated that antigen-dependent signaling induces active division of a minor lymph node resident CLL population [2,3]. These cells acquire a quiescent state upon (re-)entering the peripheral circulation, where continuous antigen-independent signaling takes over from intermittent antigen-dependent signals to extend survival of the cell before a new strong signal is received [86]. The strength of the homotypic interaction affects the survival of the CLL cell outside of the lymph nodes. Aggressive CLL cases are marked by short-lived, low avidity interactions between their BCRs, which may prove more effective in sustaining CLL cell viability compared to the long-lived, high avidity interactions observed in indolent CLL cases [83]. Importantly, heterogeneous abundance of signaling regulators like Lyn, Syk, and BTK is independent of IGHV SHM status [26, 30, 44, 76, 79], but still in some studies basal levels of abundance or phosphorylation of these genes appeared to be associated to TTFT [43,44] (Table 1). Intriguingly, the aforementioned THEMIS2 and ZAP-70 molecules have also been implicated to increase constitutive BCR activation in absence of any antigen [67, 72, 87].

Negative feedback mechanisms for BCR signaling in CLL

Currently, research on the genetic composition of the BCR and the initial downstream signaling cascade is emphasized, while less attention is given to the intricacies of negative feedback loops. Abnormal participation of inhibitory co-factors is an alternative mechanism of CLL to inflate BCR responses. This counterbalance provided by co-receptors is achieved by a universal process, in which Lyn is the central mediator of this suppression. Upon BCR stimulation, Lyn will phosphorylate the cytoplasmic tails of several inhibitory receptors, which are then able to recruit SHP-1 or SHIP-1. SHP-1 and SHIP-1 are phosphatases that dephosphorylate components of the BCR signaling cascade for negative regulation [88]. Increased expression of several receptors connected to this negative feedback mechanism is predictive of TTFT (Figure 3; Table 1).

Figure 3. Inhibitory proteins of the BCR signaling cascade in CLL that predict TTFT. Various receptors induce SHP-1/SHIP-1-mediated antagonism of the BCR signaling pathway, a negative feedback mechanism initiated by Lyn. Furthermore, the inhibitory factors SLAMF1/7, FCMR, and ENDOG/PTEN may regulate the strength of the BCR response. Created with BioRender.com. BCR: B cell receptor; CLL: chronic lymphocytic leukemia; TTFT: time-to-first-treatment.

The receptor FcγRIIb has been associated to longer TTFT [52], and this receptor is well-known to induce SHIP-1 mediated antagonism of the BCR [88].

Furthermore, CD5 has been proposed to interfere with BCR signaling in a similar manner in CLL [89–91], albeit, one paper concluded this to be false [92]. Differences in CD5-triggering have been observed in CLL [89,90], which may explain conflicting reported results. In one such report, the study cohort mostly consisted of Binet stage A patients (95%) and thus may unintentionally have been enriched for CD5 non-responsiveness, while other studies included more diverse patient groups (65% Rai stage 0–II or 47.5% Binet stage A). Notably, low CD5 surface expression is associated with shorter TTFT [53].

Diminished responses are also suggested to be dependent on the LAIR-1/SHP-1 axis [93]. Co-incidentally, patients presenting with LAIR-1 positive CLL experienced longer treatment-free survival periods from diagnosis than LAIR-1 negative CLL [54].

Although at present there is a lack of research investigating SHP-1 or SHIP-1 as prognostic biomarkers, one could hypothesize that all these receptors ultimately lead to downstream activation of SHP-1 or SHIP-1. SHP-1 and/or SHIP-1 could be considered as an integrating hub, representative for the cumulative activity exerted by various inhibitory receptors. Profiling solely the expression of these two proteins may potentially offer an efficient diagnostic approach, minimizing both labor and costs compared to the assessment of a broader array of receptors.

Having described the role of the SHP-1/SHIP-1 phosphatases as the primary negative feedback mechanism in BCR signaling, it is worth exploring other, unrelated mechanisms that have implications for TTFT (Figure 3; Table 1).

SLAMF1 and SLAMF7 attenuate the BCR pathway by competition for PHB2 in M-CLL, explaining why membrane positivity of these receptors is indicative of indolent disease [55]. Furthermore, CLL patients with elevated expression of FCMR exhibit favorable clinical outcomes [57]. FCMR influences BCR signaling in various ways, both positive and negative [94]. Yet, its predominant role appears to be inhibitory, as evidenced by the improved clinical outcomes in patients expressing FCMR.

Finally, considering the pivotal role of the BCR induced PI3K/AKT activation in CLL, it is crucial to consider ENDOG and PTEN. The ENDOG/PTEN axis governs PI3K activity, and the diminished PTEN capacity through ENDOG upregulation was indicative of a more aggressive disease course in M-CLL [56].

Moving beyond the IGHV SHM dichotomy for prognosis

The prognostic markers discussed in this review have been deliberated within the broad classification of M-CLL versus U-CLL, yet it is crucial to recognize the inherent oversimplification of this artificial dichotomy. Significant heterogeneity exists within these subgroups, particularly in the case of M-CLL. For example, although IgM expression levels generally show a trend to be lower in M-CLL compared to U-CLL, substantial diversity persists. This variability manifests itself across various facets of BCR signaling, encompassing the strength of the BCR signal received and the ensuing responsiveness. It is imperative to underscore that IGHV SHM status, despite its robustness, oversimplifies the full heterogeneity of CLL. A more nuanced understanding is achieved by considering a composite of the previously discussed BCR-signaling components. This notion is supported by a study that integrates multiple signal transducers of the BCR pathway to predict TTFT [34], which was reinforced by clustering of RNA sequencing data that distinguished CLL cases in two groups based on BCR-induced transcriptome signatures [58–60]. Notably, such comprehensive approaches outperform IGHV mutational status for risk assessment. Additionally, while single molecular biomarkers are easy interpretable for risk stratification, multiple co-occurring molecular biomarkers with independent prognostic value rapidly complicate diagnostics. However, the compound effect of a combination of molecular layers may be reduced in complexity when evaluating pre-defined profiles of signaling cascades, where all these molecular markers accumulate to achieve a similar result, i.e. dysregulation of the BCR pathway.

To further contextualize the aforementioned biomarkers within the framework of IGHV SHM status, it is worth mentioning that many studies rely on univariate survival analysis, often neglecting the confounding effect of the two subsets. While statistical differences between M-CLL and U-CLL of the measured parameter are often demonstrated within papers, these distinctions are frequently omitted as covariates in Cox regression models. Incorporating such biomarkers as covariates for risk stratification while correcting for IGHV mutational status may sometimes reveal a non-significant effect, as they may essentially align with the pre-established subsets. To ensure independent prognostic value, it is crucial to undertake multivariable analyses, a practice applied in some studies [24, 26, 31, 47, 50, 53,54]. However, an alternative approach is also observed, where researchers evaluate the biomarker value within the M-CLL and U-CLL subgroups [31, 47, 55–57]. This strategy allows the biomarker to complement established prognostic markers, further refining our understanding of the observed heterogeneity.

From theory to practice

Having reviewed all prognostic biomarkers within the BCR pathway, along with their potential pathobiological mechanisms, the next step is to discuss how to translate them into a feasible diagnostic application. It is not feasible to perform the entire variety of analytical tests discussed in this review at diagnosis, as some tests are too complex, some are too costly, and some are just not practical for routine use. Similar issues arise for the actual biomarkers to be evaluated, which cannot all be measured and analyzed. Hence, the two main considerations at stake are: what to measure, and how to measure?

When it comes to what to measure, integrating hub proteins in the BCR pathway would be a good focus. These hubs bring together signals from different parts of the pathway, making them more efficient to study than looking at each part individually. Among these hubs, the upstream integrating hub containing Lyn, Syk, BTK, and PLCγ2 is particularly promising due to its proximity to the primary defect in BCR signaling of CLL cells [34, 95].

When it comes to how to measure, flow cytometry stands out as the most effective approach. It leverages the existing infrastructure in diagnostic labs, and the current panels are expandable to include more fluorochromes. However, flow cytometry measures absolute levels of proteins, whereas BCR signaling primarily depends on sequential phosphorylation events, making the phosphorylated state of a protein a more precise indicator of pathway activity. Phosphoflow analysis holds promise, but it faces challenges like low signal resolution and transient phosphorylation events, rendering it unsuitable for routine diagnostic use at this time.

Based on the above considerations, our proposal is to merge diagnostic and prognostic testing into a single immunophenotypic test as much as possible. However, we want to emphasize that we do not support mutual exclusivity of molecular and immunophenotypic testing, but instead advocate a combination of both approaches. Great efforts have been made in advancing molecular testing as well, particularly noteworthy are the recent innovations in capture assays [96,97]. Bringing together the results of a capture assay and an immunophenotypic test could provide a thorough prognostic assessment for each patient at diagnosis, thereby personalizing the frequency of hospital visits during the watchful-waiting period. And, of course, it is essential to consider the clinical course of the patient and prioritize guidance to their specific needs.

Conclusions

It becomes evident that focusing on the BCR signaling cascade paints a canvas that is not just diagnostically intuitive, but is also functionally relevant. This approach navigates the diverse molecular landscape of CLL, offering a richer understanding of its heterogeneity beyond IGHV mutation status. Persistent endeavors to integrate these experimental studies into diagnostically feasible methodologies hold the potential to aid clinicians for better informed decision-making on expected TTFT in newly diagnosed CLL.

Acknowledgements

MYLV wrote the manuscript under supervision of AWL, MDL, and PJH with contributions from all authors, who read, commented on, and approved the final version of the manuscript.

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Funding

The author(s) reported there is no funding associated with the work featured in this article.