Key points

Introduction

In the last 60 years, the improvement of laboratory techniques has radically changed the clinical presentation of chronic lymphocytic leukemia (CLL): the percentage of cases identified through a routine blood count has constantly increased from 10% (in the 1950s) to 80% (in the late 1990s). Accordingly, they frequently present only with an asymptomatic lymphocytosis (Rai stage 0).

At the same time, thanks to the widespread availability and use of flow cytometric analysis, the detection of monoclonal B-cell populations bearing the same immunophenotypic profile of CLL has become increasingly frequent, even in the absence of a detectable lymphocytosis. This phenomenon, defined as CLL-like monoclonal B-cell lymphocytosis (MBL), has attracted great interest among CLL investigators.

MBL, as a relatively new diagnostic entity, added a new piece in the complex puzzle of CLL biology. Although the alternative definition of “B-cell lymphocytosis of uncertain significance” has been long discarded because of the potential risk of creating anxiety in affected individuals, the “uncertain significance” is definitely characteristic of MBL. More than 7 years after the first consensus criteria publication, a flurry of new information is animating the debate on the real essence of MBL and poses new and so far unanswered questions. On the one hand, it has been suggested that MBL might be a precursor state for CLL (like monoclonal gammopathy of undetermined significance for multiple myeloma). On the other hand, it has been hypothesized that the presence of monoclonal B-cell expansions could be interpreted as part of the inevitable immune system modifications (ie, restriction) occurring with aging and should not be considered a truly preneoplastic condition per se.

Several investigational studies in the last years shed some light on the biology of MBL as well as on the initial transforming events that occur in CLL. All this new information can now be used to try to understand if and how these 2 conditions are related to each other and how important it might be to distinguish them.

Introduction

In the last 60 years, the improvement of laboratory techniques has radically changed the clinical presentation of chronic lymphocytic leukemia (CLL): the percentage of cases identified through a routine blood count has constantly increased from 10% (in the 1950s) to 80% (in the late 1990s). Accordingly, they frequently present only with an asymptomatic lymphocytosis (Rai stage 0).

At the same time, thanks to the widespread availability and use of flow cytometric analysis, the detection of monoclonal B-cell populations bearing the same immunophenotypic profile of CLL has become increasingly frequent, even in the absence of a detectable lymphocytosis. This phenomenon, defined as CLL-like monoclonal B-cell lymphocytosis (MBL), has attracted great interest among CLL investigators.

MBL, as a relatively new diagnostic entity, added a new piece in the complex puzzle of CLL biology. Although the alternative definition of “B-cell lymphocytosis of uncertain significance” has been long discarded because of the potential risk of creating anxiety in affected individuals, the “uncertain significance” is definitely characteristic of MBL. More than 7 years after the first consensus criteria publication, a flurry of new information is animating the debate on the real essence of MBL and poses new and so far unanswered questions. On the one hand, it has been suggested that MBL might be a precursor state for CLL (like monoclonal gammopathy of undetermined significance for multiple myeloma). On the other hand, it has been hypothesized that the presence of monoclonal B-cell expansions could be interpreted as part of the inevitable immune system modifications (ie, restriction) occurring with aging and should not be considered a truly preneoplastic condition per se.

Several investigational studies in the last years shed some light on the biology of MBL as well as on the initial transforming events that occur in CLL. All this new information can now be used to try to understand if and how these 2 conditions are related to each other and how important it might be to distinguish them.

How to define MBL

Consensus guidelines, World Health Organization classification, and International Workshop on Chronic Lymphocytic Leukemia 2008 criteria define MBL as a clonal B-cell expansion where the B-cell count is less than 5 × 10 ⁹ /L and no symptoms or signs of lymphoproliferative disorders (ie, B symptoms, hepatosplenomegaly, lymphadenopathies) are detected. In most (>75%) cases, the immunophenotypic profile of these clonal expansions is virtually indistinguishable from that of CLL cases (ie, CD5 ⁺ , CD23 ⁺ , CD20 ^dim , surface immunoglobulin expression at low levels [sIg ^dim ]). In the remaining 25% of affected subjects, B-cell clones exhibit a different immunophenotypic profile and are classified as the following:

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  Atypical CLL MBL: CD5 ⁺ , CD20 ^bright , variable expression of CD23 (provided the lack of translocation involving the Cyclin-D1 locus);

  
  
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  CD5-negative (or non-CLL) MBL: CD5 ⁻ , without evidence of other typical markers of lymphoproliferative disorders (eg, CD10 for follicular lymphomas).

Setting a numeric threshold (<5 × 10 ⁹ /L) made it clear that finding a monoclonal B-cell population in the peripheral blood should not necessarily lead to the diagnosis of a specific disease because this type of expansion can be detected frequently in otherwise healthy subjects.

How frequent is this finding? Although in the very beginning, this condition was detected at low incidence among healthy subjects (initial studies showed a prevalence of 0.60%), more recent population studies demonstrated that CLL-like B-cell clones, in particular, are a rather common finding in the peripheral blood of otherwise healthy subjects regardless of their geographic origin or context (asymptomatic individuals performing routine blood tests for unrelated reasons as well as isolated communities involved in genetic studies). The true frequency of this phenomenon varies between 3.5% and 12% and this variation mainly depends on the sensitivity of the flow cytometric technique used, which, in turn, is related to the number of fluorochromes used (2 vs 4 vs 5 vs 8 colors), the combination of monoclonal antibodies chosen, and the number of events acquired. The more carefully one looks for it, the easier MBL can indeed be found. That notwithstanding, a plateau in MBL detection is now being approached despite the continuous technical improvement, as further gains in sensitivity apparently do not entail a proportional increase in ascertained cases. In addition, all studies showed that the prevalence of this condition progressively increases with age, being detected in 45% to 75% of people aged 90 years or older, suggesting that, although not all individuals bear a CLL-like MBL clone at any given moment of life, virtually all have the possibility of carrying a B-cell expansion when aging.

When size matters: clinical versus low-count MBL

After the definition, in 2005, of the numerical cutoff of 5 × 10 ⁹ /L to distinguish MBL and CLL, several cases, falling below the threshold that in previous times would have been labeled tout-court as CLL, started being diagnosed as MBL. Given that they are mainly identified in a clinical context (ie, during blood tests performed in asymptomatic subjects showing mild lymphocytosis), they are now called “clinical” MBL and are characterized by the presence of clonal B cells in the range of ∼0.5 to 5 × 10 ⁹ /L (median value, approximately 2.9 × 10 ⁹ /L with 95% of cases having more than 0.45 × 10 ⁹ /L clonal B cells), a concentration that can indeed be detected during routine flow cytometry testing.

In contrast, when studies were performed in the general population using more sensitive techniques, it became clear that CLL-like B cells can be frequently observed also in healthy individuals although at definitely lower concentrations, the median number of clonal B-cells being 1 per microliter (95% of cases have less than 0.056 × 10 ⁹ /L clonal B cells). These CLL-like B cells are now defined as “low-count MBL” and are the cases that account for the high frequency of MBL in the general population (for this reason, also defined as “general population MBL”).

A cumulative meta-analysis of all MBL cases, regardless of the size of the clone, highlighted a bimodal distribution of the frequency confirming that MBL is a heterogeneous category comprising dramatically different entities at least in terms of size, although with identical phenotype.

To count or not to count: problems and pitfalls in defining a B-cell threshold

All these numerical thresholds and numerical differences may seem a bit arbitrary but it has been demonstrated that the absolute B-cell count at MBL presentation is indeed the most significant independent predictive factor for disease progression. Different studies performed in the clinical setting have shown that the B-cell count, as a continuous variable, correlates with the risk of progression, progression-free survival, and overall survival. In particular, the risk of progression to overt leukemia requiring treatment in clinical MBL has been reported as being 1% to 2% and up to 4% per year, making it comparable (although significantly different) to that of Rai stage 0 CLL (∼5%). More importantly, in clinical MBL cases Kaplan-Meier curves for disease progression did not show a plateau over time, meaning that life-long periodic monitoring should be planned for these cases, similarly to what is the current practice for patients with Rai stage 0 CLL.

In contrast, the few longitudinal studies investigating the course of low-count CLL-like MBL showed that these clonal expansions tend to remain stable over time, none apparently evolving into CLL or any other lymphoproliferative disorder.

Based on this evidence, the discussion over MBL brought again to the attention of the scientific community the long-debated dilemma on the appropriate use of the term “leukemia” in CLL. It is common knowledge that CLL pursues a heterogeneous clinical course with survival ranging from months to decades. In general, about one-third of patients will never require treatment during follow-up, showing a survival identical to age-matched unaffected individuals. The remaining patients will eventually need treatment at variable times after presentation and with different degrees of aggressiveness. Similarly, it is clear since the time of the original publication by Rai and colleagues that Rai stage 0 CLL represents a melting pot, including cases bound to progress (in months or years) and patients who will never require treatment during the disease course. For this reason, in a relevant proportion of cases, mainly among those diagnosed with Rai stage 0 CLL, the “leukemia” definition carries an unnecessary psychological distress, given that some of these subjects bear an indolent nonprogressive condition that will never become clinically relevant.

Despite remarkable research efforts and a decade of studies on prognostic markers, it is not possible yet to predict at the time of diagnosis which individuals will require treatment and should therefore be intensively followed up and which will never suffer consequences from their disease and should be simply reassured and left unattended.

Based on these premises, several groups tested the possibility that by simply increasing the threshold of the B-cell count required for CLL diagnosis it would be possible to discriminate better the cases bound to progress versus the remaining ones. A value of 10 to 11 × 10 ⁹ B cells/L, proposed in different series, is indeed able to enrich for individuals at risk of progression and would help to limit the CLL diagnosis to a greater number of individuals bound to progress. That notwithstanding, the potential tradeoff of this approach is that, using a higher cutoff value, the rate of progression of those not deserving a CLL diagnosis any longer would increase as well.

Therefore the only solid conclusion that can be drawn with certainty from these studies is that no specific B-cell count cutoff alone will ever be able to segregate individuals with no risk of progression, given that these cases are strictly entangled with those with a worse prognosis.

It then becomes reasonable to postulate that dissecting specific molecular and biologic features associated with the risk of progression or with the stability of the condition might better hold the promise of identifying the subjects at risk who would deserve regular follow-ups.

Low-count MBL: the ugly duckling story

Based on numerical as well as on clinical grounds, low-count MBL seems to be an entity clearly distinct from clinical MBL/Rai stage 0 CLL, although sharing with the latter the characteristic phenotype. This intrinsic difference is also supported by the few studies on its biologic features where it has been possible to isolate and analyze the tiny populations of CLL-like B cells in the peripheral blood.

Population-based studies clearly demonstrated that, in apparent contrast with MBL definition, CLL-like clones are not necessarily monoclonal, being sometimes polyclonal/oligoclonal. This intriguing finding suggests that the acquisition of a CLL-like phenotype is not invariably linked to the occurrence of monoclonality but it might simply reflect a so far unknown functional state of activation.

Molecular studies of the immunoglobulin repertoire also supported the concept of a distinction between low-count MBL and CLL: it is well known that CLL is characterized by a preferential immunoglobulin heavy chain variable (IGHV) gene usage (including IGHV1-69, IGHV4-34, IGHV3-7, and IGHV3-23 genes), highly restricted and biased as compared with the normal adult B-cell repertoire. This peculiar set of IGHV genes is underrepresented in small-size CLL-like clones and the genes most frequently used by low-count MBL are only rarely expressed by CLL, regardless of the mutational status of the expressed IGHV genes. Moreover, in low-count MBL stereotyped receptors (ie, the presence of closely homologous, if not identical, complementarity determining region 3 sequences on immunoglobulin heavy and light chains) has been detected only in few cases, whereas they account for more than 30% of CLL cases.

Interestingly, chromosomal analysis of low-count MBL provided unexpected results. It is common knowledge that 80% of CLL cases show a restricted number of fluorescence in situ hybridization (FISH)–detected genetic lesions that carry a prognostic value : patients bearing 13q deletion have the most favorable prognosis, whereas subjects exhibiting a 17p deletion usually follow an aggressive clinical course. Unexpectedly, several studies demonstrated that low-count MBL clones do carry the same cytogenetic abnormalities, with 13q deletion being detected at a frequency similar to CLL. A few low-count MBL cases with 17p deletion have also been reported in the literature not showing sign of progression. The detection of CLL-related abnormalities in low-count MBL strongly suggests that these lesions (and in particular 13q deletion) may occur early during MBL development (if not during B-cell development) resembling other lymphoma-related aberrations (ie, t14;18) that can be found frequently also in unaffected individuals. In particular, this finding strongly supports the possibility that these aberrations are far from being causative of the disease, as corroborated by the murine model carrying a 13q14 deletion involving the DLEU2/mir15-a/16-1 cluster. This alteration, known to reduce miR15a and miR16-1 expression, may give rise to a broad spectrum of lymphoproliferative disorders including MBL, CLL, and diffuse large B-cell lymphomas but only in a low percentage of animals and after several months of life, suggesting the necessity of additional, causative hits.

Data reported from naturally occurring mouse models are in keeping with this scenario and confirm that oligoclonal and monoclonal CD5-positive B-cell expansions are a frequent phenomenon in different mouse strains with aging (eg, New Zealand Black and New Zealand White strains).

Low-count MBL: simply a sign of immunosenescence?

The late appearance of low-count MBL, in both humans and mice, together with its stability over time, led to the hypothesis that this phenomenon might be related to “immunosenescence,” the decreased immunocompetence status that occurs in elderly people. This process is characterized by B-cell compartment changes that include a reduced antibody response associated with a limited heterogeneity in isotype, antigen-binding affinity, and immunoglobulin gene use, with the frequent detection of monoclonality. T-cell alterations are also present and involve mainly the CD8 ⁺ memory T-cell subgroup and the appearance of oligo-monoclonal CD4 ⁺ CD8 ⁺ double-positive T lymphocytes. T-cell expansions have been associated with persistent latent viral infections (eg, EBV and CMV) triggering a chronic immune stimulation. Along this line of reasoning, an increased frequency of MBL clones has been demonstrated in HCV-infected patients, irrespective of age. B-cell clonal expansions mainly with atypical CLL and CLL phenotype can be observed in about 30% of hepatitis C ⁺ subjects and reach a peak of almost 40% in advanced hepatic disease.

That CLL-like MBL clones might occur in the context of a diffuse modification of the immune system is a working hypothesis supported by different lines of evidence. Clonal T-cell expansions, especially within the double-positive CD4 ⁺ CD8 ⁺ subgroup, have been detected with increased frequency in population screening CLL-like MBL cases. These T-cell clones showed a preferential usage of specific T-cell receptors (TRBV2 and TRBV8), suggesting the fascinating (but still unproven) hypothesis that the occurrence of B-cell and T-cell proliferations may be triggered by common antigenic stimuli. In particular, it has been recently reported that MBL detected in individuals with normal lymphocyte counts is associated with a decrease in immature and naive normal circulating B cells combined to T-cell subset dysregulation. These alterations, potentially leading to an impaired immunosurveillance, become more evident as the MBL cell count increases.

Is the distinction between clinical MBL and Rai stage 0 CLL meaningful?

In contrast to low-count MBL, clinical MBL seems to be strictly connected to CLL in terms of risk of progression (and indeed all CLL are always preceded by an MBL stage). One should also remember that before the publication of International Workshop on Chronic Lymphocytic Leukemia 2008 diagnostic criteria, a great part of clinical MBL were indeed diagnosed until that moment as Rai stage 0 CLL. That notwithstanding, it is now known that both conditions are a mixture of real patients whose life expectancy will be affected by the disease but also of individuals who will never develop clinical signs and symptoms. Therefore, for a clinician it would be much more crucial to know how to identify subjects bound to progress regardless of the name label rather than to make a numerical distinction that does not exclude the need in either case of prolonged follow-ups.

Several studies have aimed at dissecting differences and/or similarities between these 2 conditions in addition to the numerical value and several factors deemed to be potentially involved have been actively investigated.

It is well known that genetic factors play a relevant role in CLL occurrence and evolution, as demonstrated by the fact that a family history of CLL or other lymphoproliferative disorder is a well-defined risk factor for developing CLL. Interestingly, studies performed in relatives of familial CLL patients (ie, a family where at least 2 members are affected by CLL) demonstrated that MBL is detected at increased frequency in these subjects, regardless of their age. The overall risk of detection of CLL-like MBL in CLL families is 17 times increased in individuals less than 40 years old, an age range where MBL is almost undetectable in the general population. The higher incidence of MBL among relatives of CLL subjects is reported also in the sporadic setting whereby first-degree relatives have an MBL prevalence similar to that found in familial CLL relatives and definitely higher than that expected in the general population, again suggesting the MBL and CLL are tightly connected. The specific genes involved in this process are yet to be defined, although some studies in CLL and MBL subjects showed a similar increased frequency of individual single nucleotide polymorphisms located in genes acting as regulators of B-cell development.

Several other genetic and molecular features have been investigated in clinical MBL and Rai stage 0 CLL but none seem to be useful in differentiating these 2 conditions. Studies comparing the distribution of IGHV gene mutation, CD38, and ZAP70 expression (all consolidated prognostic factors in CLL ) were not able to detect any significant difference between clinical MBL and Rai stage 0 CLL. FISH—detected abnormalities were also comparable in most published studies ( Table 1 ). More recently, genome-wide sequencing studies identified relevant gene mutations, including NOTCH1, SF3B1, and BIRC3, associated with advanced stages of CLL and risk of transformation. As these mutations appear in low frequency in CLL cases at diagnosis (NOTCH1 mutation as low as 4%, SF3B1 mutation as low as 5%, BIRC3 mutation as low as 4% ), it is not unexpected that the frequency of NOTCH1, SF3B1, and BIRC3 mutations in clinical MBL cohorts turned out to be particularly low (3.2%, 1.5%, and 0%, respectively), therefore again not helping in narrowing down the patients at risk of progression.

Table 1

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