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Monoclonal B cell lymphocytosis and “in situ” lymphoma

Seminars in Cancer Biology, pages 3 - 14

Abstract

The finding of monoclonal B-cell populations not fulfilling criteria for a lymphoid malignancy has given great impulse to study mechanisms involved in lymphomagenesis and factors responsible for the transition from B-cell precursor states to overt lymphoproliferative disorders. Monoclonal B cell expansions were initially recognized in peripheral blood of otherwise healthy subjects (thus defined monoclonal B-cell lymphocytosis, MBL) and in most cases share the immunophenotypic profile of chronic lymphocytic leukemia (CLL). The clinical relevance of this phenomenon is different according to B-cell count: high-count MBL is considered a preneoplastic condition and progresses to CLL requiring treatment at a rate of 1–2% per year, while low-count MBL, though persisting over time, has not shown a clinical correlation with frank leukemia so far. MBL other than CLL-like represent a minority of cases and are ill-defined entities for which clinical and biological information is still scanty. In situ follicular lymphoma (FL) and mantle cell lymphoma (MCL) are characterized by the localization of atypical lymphoid cells, carrying t(14;18)(q32;q21) or t(11;14)(q13;q32), only in the germinal centers and mantle zones respectively, where their normal counterparts are localized. The localization of these cells indicates that germinal centers or mantle zones provide appropriate microenvironments for cells carrying these oncogenic alterations to survive or proliferate. The progression of these lesions to overt lymphomas occurs rarely and may require the accumulation of additional genetic events. Individuals with these lymphoid proliferations should be managed with caution.

Keywords: Monoclonal B cell lymphocytosis, Chronic lymphocytic leukemia, t(14;18)(q32;q21), t(11;14)(q13;q32), In situ lymphoma, Follicular lymphoma, Mantle cell lymphoma.

1. Introduction

The presence of B-cell premalignant states have been recently recognized in the general population but factors involved in determining progression into overt malignancy are far from being clarified. After the long-standing example of monoclonal gammopathy of undetermined significance (MGUS), a well-recognized precursor state for multiple myeloma [1] , at least two different premalignant states have been intensively investigated in the last years, i.e. monoclonal B-cell lymphocytosis (MBL) and “in situ” lymphomas, in peripheral blood and lymphoid tissues, respectively. These conditions have given great impulse to study mechanisms involved in lymphomagenesis and factors responsible for the transition from B-cell precursor states to overt lymphoproliferative disorders.

2. Monoclonal B-cell lymphocytosis

The presence of clonal B-cell populations in the peripheral blood of otherwise healthy subjects has been often reported in the literature but in the last decade, thanks to a widespread technological improvement, the detection of clonal B-cell populations not fulfilling criteria for a lymphoproliferative disorder has become a frequent finding also during routine diagnostic work-up.

An international panel of experts in 2005 defined this condition as “monoclonal B-cell lymphocytosis” and proposed formal criteria for the diagnosis, based on a B-cell count less than 5 × 109/l in the absence of signs and symptoms of an overt lymphoproliferative disease [2] . The threshold of 5 × 109 B lymphocytes/l was adopted in the new IWCLL 2008 [3] criteria and WHO classification for chronic lymphocytic leukemia (CLL) diagnosis, replacing the previous cut-off based on the absolute lymphocyte count [4] .

According to the immunophenotypic profile MBL cases were further classified as [2] and [5]:

  • CLL-like MBL: sharing the characteristic immunophenotype of CLL, characterized by CD19 and CD5 concomitant expression, low levels of surface immunoglobulin (sIg), dim CD20 expression, CD23 positivity;
  • Atypical CLL MBL: expressing CD5 together with bright CD20 expression and/or lack of CD23 expression; in this case extensive lymphoma work-up, paying particular attention to FISH analysis for t(11;14), is required to exclude mantle cell lymphoma (MCL) diagnosis;
  • CD5 negative (or Non-CLL) MBL: lacking CD5 expression. It is usually devoid also of CD10 expression and other lymphoma-specific markers, maybe resembling a precursor condition for marginal-zone lymphomas (MZL). One should also consider that a large proportion of leukemic MCL with small cell variant and indolent behavior are CD5-negative [6] and [7]. Therefore, a FISH analysis for the t(11;14) may be appropriate also for individuals carrying a CD5-negative MBL.

Monoclonal B-cell expansions similar to CLL cells are the most frequently detected (roughly 75% of MBL cases) and have been extensively studied in the most recent years with some unexpected and exciting findings.

3. CLL-like MBL

Monoclonal CLL-like B-cell expansions were first recognized more than 20 years ago in population-based studies analyzing the presence of abnormal B-cell populations as a biomarker of environmental exposure in people living around hazardous waste sites. Initially, the clinical relevance of such finding was considered limited as the overall prevalence of MBL was estimated to be very low (11/1926 individuals, i.e. 0.6%) [8] and a low prevalence was confirmed by studies analyzing blood donor samples where only 7 out of 5141 blood donors (0.14%) were found to carry monoclonal B-cell expansions [9] . Novel studies performed in recent years using high-sensitivity flow cytometry techniques demonstrated that CLL-like expansions are a frequent and consistent phenomenon and can be detected in different geographical and demographical settings [10] and [11]. Thanks to these technical improvements, it became evident that the prevalence of CLL-like expansions was previously underestimated as it might range between 3.5 up to 12% of healthy individuals older than 40 years [10], [11], [12], [13], and [14]. Differences in reported prevalence are explained by different sensitivity of flow cytometric approaches, depending on types and combinations of fluorochromes as well as on the number of events acquired [15] . Epidemiological studies showed that MBL prevalence is higher among male subjects and increases with aging, being detected in up to 50–75% of people >90 years [12], [14], and [16]. In addition, the frequency of MBL is significantly higher in first-degree relatives of CLL patients [17] and [18], in line with the well-known familial predisposition of CLL and of other indolent lymphoproliferative disorders.

Recent meta-analysis based on series reported in the literature clearly pointed out that the term MBL is applied to different conditions, that belong to different settings and carry a different likelihood of representing a true pre-leukemic state [19] . Along this line it has been clearly defined that at least two different entities may be recognized, based on the number of clonal B cells [5] and [19]. Cumulative analysis of all CLL-like expansions from different international series, showed a bimodal distribution in clonal B-cell count with a lower peek between 0.1 and 50 clonal B cells per μl, and a second upper peak between 500 and 5000 clonal B cells per μl, with only few cases lying in between [19] . The lower peak condition, now named low-count MBL, is characterized by a very low concentration of CLL-like B cells in the peripheral blood (median number of clonal B cells being only 0.001 × 109/l) and it is usually found in population screening studies performed for research purposes (therefore previously known as population-screening MBL). This condition does not have a significant clinical correlation with a frank leukemia as the risk of progression into CLL seems to be negligible, if any [13] .

The second peak corresponds to the so called high-count MBL, usually detected in the clinical setting (and therefore also defined as “clinical MBL”), where clonal B-cell count reaches a median value of 2.9 × 109 cells/l and 95% of cases have more than 0.45 clonal B cells/l. This condition is usually associated with the presence of lymphocytosis and carries a risk of progression into CLL requiring treatment of 1–2% per year [20], [21], [22], [23], [24], and [25].

3.1. Multistep development of CLL

How we get to a full-blown CLL starting from a normal B lymphocyte is still a matter of debate. Essential ingredients likely include genetic lesions and microenvironmental stimuli, in particular B-cell receptor (BCR) triggering by yet unrecognized self or foreign antigens. Though at the beginning the MBL definition seemed to pose more questions than answers, recent data derived from population-based and clinical series clarified a number of aspects that may help to better define at least the initial stages of CLL.

To this aim, the first question we should ask is “Should we consider MBL a truly preneoplastic condition?” Preneoplastic conditions are usually defined as an early step in tumor development consisting of genetically and phenotypically altered cells exhibiting a higher risk of malignant evolution than normal cells. In this framework a preneoplastic condition is usually an early finding in tumor development that can be used as surrogate marker for tumor predisposition and in some cases can represent a therapeutic target to prevent tumor development.

A nationwide prospective population-based cancer screening trial in US clearly demonstrated that virtually all cases of CLL are preceded by MBL, thus confirming its preneoplastic nature [26] . Samples collected from 6 months to 6 years before the development of clinically recognized leukemia showed the presence of pre-diagnostic monoclonality among B-cells in all cases, with unmutated as well as mutated immunoglobulin heavy variable (IGHV) genes (see next). This finding was independent from clinical stage at diagnosis, as the presence of prediagnostic clones was detected in cases with low (stage 0 or I according to Rai classification) as well as high (stage II or III) tumor burden at diagnosis.

If it is undeniable that MBL always precedes CLL occurrence, it should be considered that not all MBL cases are bound to progress into CLL requiring treatment as high-count CLL-like MBL progresses into CLL requiring treatment only at a rate of 1–2% per year in most published series [20], [21], [23], and [24]. Thus, MBL cannot be considered a surrogate marker for leukemia predisposition tout court and additional factors responsible for the transition are yet to be discovered and validated ( Fig. 1 ). Even more elusive remain the essence of low-count MBL, that lacking overall the capacity to progress into frank CLL, seems to play a minimal if any role as preneoplastic condition, and claim to be a bird of different feather ( Fig. 1 ).

gr1

Fig. 1 Hypothetical model for molecular pathogenesis of chronic lymphocytic leukemia (CLL). It has not been clearly defined if the first hit in the development of clonal CLL-like expansions relies on external stimuli or acquired genetic abnormalities. Once the development of high-count MBL occurs, this condition progresses to full-blown CLL at a rate of 1–2% per year. Low-count MBL and CLL share the same immunophenotypic profile and genetic alterations (in particular 13q deletion) but progression from low-count MBL to CLL has not been demonstrated. source: Flow cytometry plots: kind courtesy of Dr. Claudia Fazi; FISH imaging: kind courtesy of Dr. Lorenza Pecciarini.

To this purpose, immunogenetic studies and the analysis of IGHV gene repertoire gave us precious hints in understanding these early events. CLL is characterized by a restricted IGHV gene repertoire, showing a preferential usage of specific IGHV genes (mainly represented by IGHV1-69, IGHV4-34, IGHV3-7 and IGHV3-23) [27] and [28]. More importantly, it has long been demonstrated that CLL cases can be categorized according to the IGHV gene mutation status as mutated (germ-line identity less than 98%, found in 65–75% of newly diagnosed patients) or unmutated (germ-line identity 98% or more, 25–35% of cases). Along with its biological significance, the IGHV gene mutational status carries a prognostic relevance, as unmutated cases tend to follow a dismal clinical course [29] and [30]. In addition, more than 30% of cases carry highly similar, quasi-identical immunoglobulin receptors (so called stereotyped BCR) [31] and [32]. The apparently non-stochastic expression of specific stereotyped BCRs, strongly supports the likelihood of antigen-driven selection in CLL clones. Several studies demonstrated that in clinical MBL the immunoglobulin repertoire and the frequency of BCR stereotypy (around 30%) are very similar to those of Rai Stage 0 CLL cases, while low-count MBL cases carry a different immunoglobulin repertoire and very rarely express stereotyped BCR [33] . IGHV genes usually expressed at higher frequency among elderly people (e.g. IGHV4-59/61) are in parallel overrepresented in low-count MBL [12] and [33]. This finding is in keeping with the idea that IGHV repertoire in low-count MBL may represent a form of restriction due to age and that this condition may be more related to immune senescence rather than to leukemogenesis. Similar mechanisms have been previously proven in peripheral blood T-cell subpopulations where clonal expansions associated with TCR repertoire restriction, especially in the CD8+ and CD4+/CD8+ double positive compartments, were found at increased frequency with aging [34] and [35]. The association between the presence of T-cell clonal expansions and chronic latent viral infections (e.g. EBV and/or CMV) may be considered an indirect evidence of TCR stimulation by specific antigens at the basis of this phenomenon [36] . Along the same line an increased prevalence of MBL (both CLL-like and non-CLL-like MBL) has been found in HCV-infected subjects [37] .

The scenario starts getting even more complicated when we focus our attention on the role of genetic aberrations. At variance with other lymphoproliferative disorders where a predisposing or causative genetic lesion has been recognized, CLL is not associated with a unique cytogenetic alteration. Yet, in more than 80% of CLL cases FISH analysis shows the presence of at least one out of 4 most frequent genomic aberrations, including del(13q), del(11q) and del(17p) and trisomy 12 [38] . These genetic alterations have been proven to play a prognostic role in CLL clinical course, as patients with del(13q) > normal karyotype > trisomy 12 > del(11q) > del(17p) have a progressively worsening disease course. In terms of molecular pathogenesis, the unfavourable clinical outcome of patients with del(17p) is explained by the loss of TP53, a well-characterized tumor-suppressor gene, frequently undergoing inactivating mutations on the other allele as in most human cancers. On the other side the favorable prognosis of patients bearing del(13q) has been recently linked to specific microRNAs (miR15a/16-1) loss, acting as tumor suppressor genes. A recently published mouse model carrying DLEU2/miR-15a/16-1 deletion has been shown to develop low-penetrance indolent B-cell lymphoproliferative disorders resembling the whole spectrum of CLL associated phenotype (from MBL through CLL to diffuse large B-cell lymphoma, DLBCL) [39] . Somehow unexpectedly when the presence of genomic aberrations in clinical and low count MBL cases has been tested, all these alterations (including the one associated with worse clinical outcome) were detected. In particular del(13q) was detected even in around 50% of low-count MBL cases, suggesting an association with the acquisition of a CLL-phenotype rather than with a true leukemic condition. Along the same line of reasoning, the presence of t(14;18), the hallmark of follicular lymphoma, has been found in more than 50% of otherwise healthy individuals, not necessarily implicating a lymphoma diagnosis neither a predisposition to lymphoma development (see Section 6.1 ).

Somehow different is the situation when we consider the novel genetic mutations as recently discovered by next generation sequencing studies of CLL [40] . MYD88 [40] , NOTCH1[40] and [41], SF3B1[42] and [43] and BIRC3 [44] were the most frequently detected recurrent mutations. The proportion of CLL cases showing these alterations seems to be quite low at diagnosis and most of these genes have been associated with chemoresistance and disease transformation occurring at later stage of CLL course. Accordingly, mutations of these genes are a very infrequent finding if any in clinical MBL cases so far analyzed thereby not helping in defining the early steps of both MBL and CLL development [44], [45], and [46].

These evidences strongly support the notion that we are making progress but we have not yet identified univocal molecular and clinical signatures able to discriminate cases with progression to overt disease and future research efforts should be focused on this goal ( Fig. 1 ).

3.2. Pathological findings of CLL-like MBL

CLL holds a unique position among lymphoproliferative disorders as histological evaluation in the diagnostic work-up is not necessarily required. Thus, for CLL patients histological data is scanty and even less for MBL subjects as bone marrow examination should be performed only before starting treatment, according to the revised guidelines [3] , and lymph node biopsy is mainly obtained during disease course to exclude transformation to diffuse large B cell lymphoma (DLBCL).

A notable exception exists when the tumor burden is mainly localized in lymph nodes with limited PB involvement (<5 × 109/l B cells) implying a diagnosis of small lymphocytic lymphoma (SLL). Nevertheless, SLL is less common than CLL, representing only 15% of CLL/SLL cases [47] and 6% of non-Hodgkin lymphomas [48] . There are no clear distinction criteria between CLL and SLL, as they are considered to represent different clinical manifestations of the same disease.

Invasion of primary and secondary lymphoid tissues by CLL cells disrupts the normal tissue architecture and function. In most cases spleen and lymph nodes show a diffuse infiltration by CLL cells, while bone marrow involvement is associated with variable (interstitial, nodular and/or diffuse) patterns.

Lymph node (and, to a lesser extent, bone marrow) involvement by CLL is characterized by the presence of pale areas on a dark background of small cells. These areas, so called “proliferation centers” or “pseudofollicles”, contain clusters of prolymphocytes and paraimmunoblasts, mixed with T cells (in most cases CD4 positive) and dendritic cells [49] . CLL cells located in proliferation centers show increased expression of proliferation-associated markers, like Ki-67 and CD71, and anti-apoptotic proteins, like survivin and BCL2 [50] , and are thought to represent the proliferative compartment that fuels the disease [51] .

Recent gene expression profile studies comparing gene expression in CLL lymphocytes isolated from peripheral blood (PB), lymph node (LN) and bone marrow showed that CLL cells obtained from LN specimens upregulate the expression of more than 100 genes usually expressed in response to BCR triggering and related to NFkB signaling pathway [52] . These findings further support the hypothesis that the most relevant events in CLL pathogenesis take place in tissue microenvironment where antigenic stimulation occurs and PB CLL cells, though being more easily accessible, are not likely the best candidates to clarify CLL pathogenesis.

Recently, histopathological studies performed on lymph node biopsies documented that a tissue-equivalent of MBL might be also detected in lymph node specimens with preserved architecture without fulfilling criteria for SLL. These were represented by subtle follicular and perifollicular involvement by CLL-like cells surrounding the non-neoplastic germinal center with focal proliferation centers [53] .

Nearly half of these cases were incidentally found in lymph node specimens removed for reasons other than enlarged lymphoadenopathies and mainly represented by solid tumor staging. Interestingly enough, only 5 out of 36 subjects (13%) with this “nodal MBL” [54] progressed to CLL/SLL requiring treatment, though one third (12/36) in the end fulfilled CLL/SLL diagnostic criteria. The only factors associated with progression risk were the presence of proliferation centers and the detection of any lymph node of at least 1.5 cm on radiological examinations.

3.3. Clinical implications

Based on all these evidences, MBL as a whole is actually a mixture of different entities that share a common immunophenotypic profile but follow different clinical behaviors. From a clinical standpoint the most relevant and yet unanswered question is how to distinguish those cases who will eventually progress from those who will remain unchanged. This distinction implies the possibility to select the subjects who deserve regular follow-ups (and maybe in the future preventive therapy) as bound to develop CLL requiring treatment, while reassuring the others, carrying only a clinically irrelevant laboratory abnormality that their condition will never progress to overt disease. The chance to operate this distinction would be particularly relevant in the context of an ever-aging population, where the prevalence of MBL would increase, with an increase of the costs for the health-care systems that in most cases will be unnecessary.

Biological factors implicated in CLL pathogenesis and frequently applied to define CLL prognosis (IGHV gene mutational status, CD38 and ZAP70 expression, cytogenetic aberrations) did not show different distribution among clinical MBL and early stage CLL cases and were not useful in improving progression risk definition [20] and [21]. The absolute clonal B-cell count at presentation has been demonstrated so far to be the most significant factor in predicting evolution from MBL to CLL. In clinical series, clonal B-cell count below 1.2–1.9 × 109/l were able to predict a lymphocyte count stable over time while B-cell counts above 3.7–4.0 × 109/l were correlated with a tendency to B-cell concentration increase [20] and [55].

Different thresholds have been proposed in order to improve the ascertainment of clinically significant cases. In several recently published series a threshold of about 10–11 × 109 B cells/l was able to better identify progressive cases [22], [25], and [56]. The rate of progression in subjects with a B-cell count >10–11 × 109/l reached the value of 5–8% per year but a relevant drawback of this approach was that the risk of progression in the “newly-defined” MBL reached the value of 2.5%, claiming the need for regular follow-up also in this category of individuals, without any practical advantage in terms of clinical management. These results clearly demonstrated that no solid conclusions can be drawn based only on B-cell count and we should aim our research efforts at identifying molecular features responsible for stability or risk of progression.

Another relevant issue that has been raised relates to the need of performing high sensitivity imaging technique (like CT scan) as part of diagnostic work-up in clinical MBL cases. Current guidelines discourage the use of CT scan as initial work-up for CLL in general and for CLL-like MBL in particular, though a number of studies showed that an under-recognized grey zone between SLL and clinical MBL may exist, where, in the presence of <5 × 109/l B cells, enlarged lymph nodes would indicate an SLL diagnosis [53] . This finding cast doubts on the fact that physical examination could not be sufficient as initial workup of clinical MBL patients. Unexpectedly, recent series confirmed that only a very low percentage (about 6%) of clinical MBL cases, had lymphoadenopathies and/or hepatosplenomegaly as detected by imaging studies (chest X-ray and abdominal ultrasound) performed at diagnosis, thereby confirming the current indications [25] .

At present, the clinical management of MBL cases is based on expert panel suggestions and derives from the results of few population-based and clinical series followed-up over time. It seems extremely reasonable that low-count MBLs whose risk of progression into CLL requiring treatment is negligible, if any, should not be candidates to clinical follow-up except for research purposes. This is essentially based on the fact that none of the low-count MBL re-evaluated after a median follow-up of almost 3 years developed a lymphoproliferative disorder and, though the persistence of CLL-like B-cell clones has been confirmed, clonal B-cell counts tended to remain stable [13] .

In contrast, as there are no biological or clinical factors able to differentiate individuals with clinical MBL at higher risk of progression, all these cases should undergo regular follow-up as for MGUS cases ( Table 1 ). Yet, this category of patients remain too wide as recent studies have demonstrated that the life expectancy in clinical MBL subjects is overall not statistically different from that of age- and gender-matched general population, in line with its classification as pre-malignant condition [57] . That notwithstanding, a subgroup of clinical MBL, especially expressing higher levels of CD38 on cell surface showed a reduced life expectancy in comparison to normal controls [57] . This evidence indicate the impellent need of finding reliable and reproducible biological factors that could be of help in better characterizing prognosis in this group of patients, relieving the majority from unnecessary clinical attention and psychological distress.

Table 1 Clinical course (at least one year follow-up) of high-count and low-count CLL-like MBL.

Diagnosis Case number Sex (M:F) Median age (range) Observation period median years (range) Case number with development of CLL requiring treatment Time to first treatment median years (range) Reference
High-count MBL 185 9.3:10 71 (39–99) 6.7 (0.2–11.8) 13/185 (7%) 4.0 (1.1–10.1) [20]
  302 13.8:10 69 (34–93) 1.5 (0.0–8.1) 7/302 (2.3%) Not reached [21]
  123 9.5:10 68 (59–75) a 3.5 (n.r.–n.r.) 19/123 (15.4%) Not reached [55]
  124 9.3:10 65 (32–100) 3.9 (0.2–10.0) 19/124 (15.3%) Not reached [56]
  184 11.9:10 64 (56–70) a 3.8 (0.0–25.5) 24/182 (13.2%) Not reached [25]
Low-count MBL 54 18.4:10 66 (40–92) 2.8 (0.9–4.2) 0/54 (0%) n.a. [13]

a 25th–75th percentiles.

MBL, monoclonal B cell lymphocytosis; CLL, chronic lymphocytic leukemia; M, male; F, female, n.r., not reported; n.a., not applicable.

4. MBL other than CLL-like

MBL carrying an immunophenotypic profile different from CLL are ill-defined entities. Provided that they are less frequent than CLL-like MBL, clinical data is scantier and derives from few series and case reports.

As previously mentioned, based on their immunophenotypic profile, MBL non resembling CLL phenotype can be further divided in atypical CLL (CD5 positive, CD20 bright and/or lacking CD23 expression) and CD5 negative (non-CLL) MBL [5] . In general population studies the frequency of these conditions is less affected by aging in comparison to CLL-like MBL and the prevalence ranges between 1 and 2.5% [11], [12], and [13]. It has been proposed that this low frequency may be at least partially explained by ascertainment bias, especially for the CD5 negative subgroup. As the phenotypic features of these conditions are very heterogeneous and there are no specific immunophenotypic markers to differentiate the abnormal populations, current flow cytometry panels are able to detect CD5 negative clonal populations only if they represent the largest part of B cells in the peripheral blood [58] .

As for CLL-like MBL, atypical CLL and CD5 negative MBL detected in population studies and in the clinical setting seem to carry a different clinical and biological significance. In our experience, when we evaluated the persistence of monoclonal B-cell expansions detected in the general population after almost 3 years of follow-up, around 55% and 35% of atypical CLL and CD5 negative clones, respectively, have disappeared [13] . These results, though being discordant with those reported from a previous study with a shorter follow-up length [58] , suggested that these kinds of expansions might be transient and self-limiting. Along the same line, our and other general population studies showed that more than one clonal B-cell expansion with atypical CLL or non-CLL characteristics is frequently detected in the same subject [12], [13], and [58]. Some series reported that more than 30% of cases carried two different clones [58] .

In addition to that, as previously mentioned, when the prevalence of MBL was investigated in HCV infected patients, all MBL subgroups were detected at increased frequency in comparison to the general population [37] . The increase in atypical CLL MBL prevalence was particularly impressive, as its frequency was ten times higher than that reported in age-matched healthy individuals. Based on these findings it has been proposed that MBL other than CLL-like, at least in the general population setting, can be interpreted as “reactive” monoclonal expansions triggered by immune stimulation.

On the other side immunophenotypic, cytogenetic and molecular studies performed on atypical CLL and CD5 negative MBL with lymphocytosis, diagnosed in a clinical setting, gave inconclusive results. In this situation, the aim of defining the likely malignant counterpart of atypical CLL and CD5 negative MBL, if any, was particularly challenging considering that indolent lymphoproliferative disorders other than CLL do not exhibit a distinctive immunophenotypic profile and the diagnosis is usually based on histopathological findings.

As the detection of CD10+ clones in the peripheral blood is rather infrequent, marginal-zone lymphoma (MZL) and, to a lesser extent, lymphoplasmacytic lymphoma (LPL) remain the most likely malignant counterpart for MBL other than CLL-like, especially those CD5 negative, according to the immunophenotypic and genetic features. In studies performed mainly on CD5 negative MBL, cytogenetic alterations detected by FISH are reminiscent of the cytogenetic profile found in MZL, with a higher frequency of aberrations in 7q region [59] . In particular del(7)(q31-34) and t(2;7) have been described in a small series of CD5 negative MBL and both alterations were previously found to be associated with splenic marginal-zone lymphoma (SMZL) [60] . The presence of isochromosome 17q and translocations involving the (14)(q32) region are other cytogenetic alterations reported in both CD5 negative MBL and MZL cases.

In contrast, a recent immunogenetic analysis gave more controversial results [61] . CD5 negative MBL cases bear a significantly higher somatic hypermutation load (SHM) compared to SMZL and immunoglobulin genes were shown to be mutated (according to the cut-off derived from CLL, less than 98% identity to germ-line) in around 85% of CD5 negative MBL. Though initial studies showed no preferential usage of specific IGHV genes [59] , more recent reports found an overrepresentation of IGHV4-34 gene among MBL cases along with a very low frequency (only 1 among 63 cases, 1.6%) of IGHV1-2 gene use [61] that is instead preferentially used in almost 30% of SMZL cases [62] .

Though the clinical significance of MBL other than CLL-like is far from being clarified, the major challenge from a clinical point of view is to distinguish these conditions from the leukemic phase of a lymphoproliferative disorder. In particular, in case of atypical CLL MBL, due to its close resemblance with mantle cell lymphoma (MCL), the consensus panel on MBL strongly recommends that every effort should be made to rule out MCL diagnosis. To this aim FISH testing with probe for t(11;14) is advised and a more intensive diagnostic work-up including bone marrow biopsy and CT scan should be accomplished. The few individuals with phenotypic and cytogenetic profile suggestive of MCL but without conclusive staging evaluation (no detectable lymphoadenopathies nor bone marrow involvement), should be more closely followed-up and repeat a CT scan at least every 6 months in order to detect early full-blown lymphoma [5] .

For CD5 negative MBL, the clinical management is based on the results of small series with longer follow-up and expert opinions. The diagnostic work-up also includes baseline CT scan evaluating chest-abdomen and pelvis and bone marrow biopsy. As limited information is available about the clinical course of these clonal expansions, it seems reasonable to follow-up patients according to the clinical features of the lymphoproliferative disorder they are at risk of developing. For this reason, non-CLL or atypical CLL MBL, where MCL features have been excluded, can be followed with less-intensive work-up, as, in the few subjects who will progress into an overt lymphoma, the disease usually can be categorized as indolent. Accordingly, recent reports showed that, after almost 5 years of follow-up, 54/71 (76%) of subjects with atypical CLL or CD5 negative MBL with lymphocytosis but lacking t(11;14) did not experience signs or symptoms of progression to an overt lymphoma. Among the 17 progressive cases 13 cases developed splenomegaly, therefore being considered as affected by SMZL/SLLU (splenic lymphoma/leukemia unclassifiable), two subjects developed lymphoadenopathies (therefore classified as possible nodal MZL), one was affected by gastric MALT lymphoma and one was diagnosed with DLBCL of the skin [61] .

5. “In situ” lymphomas

The term “in situ” in the context of neoplastic lesions usually refers to atypical cell proliferations that have acquired certain features of a neoplastic process but remain localized within the histological compartment where their normal counterparts are located. These lesions are considered an early step in the neoplastic transformation with very limited malignant potential. This term has been used classically for the diagnosis of intraepithelial neoplasms (e.g. in situ carcinoma of uterine cervix) where the basal membrane constitutes a topographic and functional barrier but it has been difficult to apply in the lymphoid system in which the cells physiologically circulate and colonize different tissues and histological compartments. However, recent studies [63] and [64] have recognized in situ lymphoid proliferations with molecular and phenotypic features of well characterized lymphomas such as follicular and mantle cell lymphomas (FL, MCL) but restricted to the germinal center and mantle zone of the lymphoid follicle, respectively. In both lesions, the atypical cell proliferation tends to substitute the normal cells in these compartments without disturbing their respective architecture. These lesions have been identified because germinal centers and follicular mantle zones are distinctive histologic structures and the respective normal cell counterparts relatively well defined [65] . The pathogenic significance of these lesions is not well understood and their diagnostic criteria and clinical implications represent relevant challenges in the clinical practice.

6. In situ follicular lymphoma

In situ FL was initially recognized a decade ago by Cong et al. [64] as the presence of atypical B-cells with a strong expression of BCL2 and CD10 in the germinal centers of lymphoid follicles of otherwise reactive lymph nodes. This atypical phenotype was associated with the presence of the translocation t(14;18)(q32;q21), the genetic hallmark of FL. However, contrary to an overt FL, these cells were restricted to germinal centers without evidences of dissemination to the interfollicular areas or effacement of the normal architecture of the lymph node. Some of these cases were associated with an overt lymphoma in other territories but most of the patients did not have evidences of simultaneous or subsequent disseminated FL after a long follow-up. These findings suggested that the lesion could correspond to an early step in the development of follicular lymphoma and the term “in situ FL” was proposed.

6.1. Multistep development of FL

The recognition of in situ FL has emphasized the perspective of the pathogenesis of FL as a multistep process in which this lesion could represent an intermediate stage between the acquisition of the initial translocation t(14;18) in the bone marrow and the development of an overt disseminated lymphoma. The t(14;18) translocation is considered the initial oncogenic event in FL and it seems to occur in the pro-B/pre-B cells in bone marrow ( Fig. 2 ). This chromosomal translocation juxtaposes BCL2 gene to immunoglobulin heavy chain gene (IGH), resulting in constitutive overexpression of BCL2 protein [65] . Breakpoints of BCL2 are concentrated at major cluster regions, intermediate cluster regions and minor cluster regions. These breakpoints are mainly distributed near the dinucleotide sequence CpG indicating cooperative effects by RAG endonuclease and activation-induced cytidine deaminase (AID) [66] ( Fig. 2 ).

gr2

Fig. 2 Schema of molecular pathogenesis of follicular lymphoma (FL) and mantle cell lymphoma (MCL) and their association with microenvironments. The characteristic genetic alterations, t(14;18)(q32;q21) and t(11;14)(q13;q32), occur in the bone marrow. Clonal B cells carrying these translocations may be detected in the peripheral blood of healthy individuals at very low levels. In situ FL or MCL are rarely identified in lymph nodes. The majority of them do not progress to overt lymphoma. Additional genetic alterations seem to contribute the development to overt lymphoma.

The acquisition of this translocation is not sufficient for the development of a FL and its oncogenic potential will be only developed in the germinal center of the lymphoid follicle. Along this line, several studies using very sensitive methods have identified circulating cells carrying the t(14;18)(q32;q21) in 50–67% of healthy individuals [67], [68], [69], [70], [71], and [72]. The prevalence of cells with this translocation is about 1 in 105 or less circulating B cells. These clones persist over long periods of time and tend to increase with aging [68] . Cell sorting methods have revealed that the phenotype of these cells is IgD+/−, CD27+, resembling memory B cells [73] . Pesticides, smoking, and age are associated with increased frequency of cell clones carrying the t(14;18) [69] and [70]. Some individuals carry several different clones with the t(14,18), although generally one of them predominates over the others. Although conceptually these clones may be considered as an initial step in the development of in situ or overt FL, clear evidences for this progression have not been documented. On the other hand, the marked differences in the prevalence of the circulating clones carrying the t(14;18) (50–67% of healthy individuals) and the low frequency of in situ FL in reactive lymphoid tissues (2–2.6%) [63], [64], and [74] support the idea that most of these clones will not progress to “in situ” FL lesions. These findings in humans are in agreement with experimental observations indicating that B cells in transgenic mice harboring the BCL2-immunoglobulin fusion gene survive longer but do not develop lymphomas [75] and [76]. On the other hand, Sakai et al. [77] analyzing B cells in Eμ-BCL2 transgenic mice proposed that inhibition of B cell differentiation might facilitate the accumulation of B-cells carrying the t(14;18) in germinal centers. These experimental results and the clinical observations support the hypothesis that germinal centers provide an appropriate microenvironment for the expansion of pre-malignant B cells carrying t(14;18). The constitutive BCL2 expression due to the t(14;18) may be a selective advantage in this microenvironment where BCL2 is physiologically down-regulated ( Fig. 2 ). However, the mechanisms that would lead to these expansions in some clones and not others are not understood.

The progression from an in situ FL lesion to an overt disseminated lymphoma may occur in certain patients but the frequency of this process seems low [64], [78], and [79]. The identification of simultaneous in situ and overt FL in other territories and the subsequent development of a disseminated FL after the diagnosis of an in situ lesion would support this progression. A recent comparative genetic analysis of an in situ FL and the synchronous overt FL has observed additional numeric chromosomal aberrations only in the overt FL indicating that in situ FL is a true premalignant lesion rather than an instance of early colonization of a disseminated FL [80] . This study also suggests that the progression of an in situ lesion to FL requires additional gene abnormalities ( Fig. 2 ).

6.2. Pathological findings of in situ FL

The identification of in situ FL is frequently an incidental finding since the lymph nodes have a preserved architecture and reactive appearance ( Fig. 3 a and b) [64] and [65]. Immunohistochemical stains may reveal strong CD10 and BCL2 expression in some cells of the germinal centers that contrasts with the weaker CD10 and negative BCL2 of reactive follicles ( Fig. 3 c and d). The BCL2 staining of these cells is usually stronger than other BCL2 positive normal cells (e.g. mantle zone B cells or T cells) ( Fig. 3 c) [81] . The atypical cells are restricted to the germinal centers without extension to inter-follicular zones and usually not all the follicles in the same lymph node are involved. The atypical cells are usually intermingled with BCL2-negative cells suggesting only a partial involvement of the germinal center ( Fig. 3 c). Some reports have identified similar in situ FL lesions in the context of progressive transformed germinal centers [82] and [83].

gr3

Fig. 3 Pathological findings of in situ FL. (a and b) Reactive lymph node with preserved architecture and hyperplastic follicles (hematoxylin and eosin, HE). (c) BCL2 staining is stronger in germinal center cells than other BCL2 positive normal cells in mantle zones. One of the germinal centers shows a partial involvement with a remaining area BCL2-negative (arrow). (d) CD10 is very strong in the germinal centers but no CD10 positive tumor cells are seen outside the germinal centers (Olympus BX51, magnification 40× for HE (a), 100× for HE (b), CD10 and BCL2).

The in situ FL lesions must be distinguished from the partial involvement of a lymph node by follicular lymphoma (PFL). The criteria for this differential diagnosis have been recently refined [78], [84], and [85]. Contrary to in situ FL, the architecture of the lymph node in PFL is only focally altered and some reactive follicles still persist. These spared follicles are usually few or even solitary whereas the affected areas have the conventional aspect of FL with interfollicular infiltration of tumor cells. Involved follicles are often larger in size and grouped together. The interface between the germinal center and mantle zone is blurred and the latter frequently attenuated. The distinction of this pattern has clinical implications. Although patients with PFL have a limited disease, usually only stage I or II, they progress to a more disseminated lymphoma more frequently than in situ FL (28% vs 4%, respectively) [78] . However, these two patterns are associated with a similar incidence of simultaneous disseminated FL elsewhere, 13% in PFL and 18% in in situ FL [78] .

6.3. Clinical implications

The clinical implications of an in situ FL lesion may be summarized in three aspects: the coexistence of a simultaneous disseminated FL, the risk of the development of subsequent overt FL, and the association with a second subtype of lymphoma. Two studies including large series of in situ FL have reported the presence of a simultaneous disseminated FL in 16–23% [78] and [79], a relative high incidence that justifies the work-out of the patients to rule-out this potential complication. To estimate the risk of progression to overt lymphoma we have reviewed 33 cases of in situ FL without simultaneous overt FL previous reported ( Table 2 ) [64], [78], [79], and [86]. We collected cases with at least one year follow-up. The age of these patients had a broad range from 23 to 85, and there were more females (20 cases) than males (13 cases). In situ FL was detected usually in only one lymph node, but in some cases the lesions could be documented in multiple lymph nodes [78] and [83]. Clinical follow-up was from 12 to 132 months, and 2 of 33 cases (6%) developed an overt FL with a time to progression of 15 and 29 months [78] and [79]. One patient [86] had atypical lymphoid cells in peripheral blood at diagnosis associated with the in situ FL lesion. However, the disease was free of progression for 11 years. This relative low incidence of evolution to overt FL has questioned the malignant potential of the in situ lesions and the term “in situ involvement by FL-like cells of uncertain significance” has been suggested [87] . The terminology for these lesions may need further discussion and consensus. Particularly, the words “uncertain significance” that, although included in the classical term “monoclonal gammopathy of uncertain significance”, they have been eluded in the more recent term, monoclonal B-cell lymphocytosis (MBL), to avoid the possible anxiety they could create in the affected patients [54] . Regardless of the term used, the low incidence of progression to overt FL supports a conservative management of these patients.

Table 2 Clinical course (at least one year follow-up) of in situ FL or MCL without overt lymphomas at diagnosis a .

Diagnosis Case number Sex (M:F) Age (median) Observation period (median, months) b Case number with development of overt FL/MCL Time to progression of overt FL/MCL (median, months) b References
In situ FL c 33 13:20 53 (23–85) 40 (12–132) 2/33 (6%) 22 (15–29) [2], [16], [17], and [24]
In situ MCL c 15 7:8 70 (29–84) 36 (12–234) 1/15 (7%) 48 (48–48) [1], [32], and [45]

a “Overt lymphoma” means overt FL or overt MCL for in situ FL or in situ MCL, respectively. Patients with circulating atypical B cells in peripheral blood were not regarded as the overt lymphoma.

b Some of original reports used “year” as a unit of measurement. We re-calculated as “months” for easy comparison.

c Eight patients of in situ FL and three patients of in situ MCL were accompanied by other type B cell lymphomas.

FL, follicular lymphoma; MCL, mantle cell lymphoma; M, male; F, female.

Some in situ FL are associated with other subtypes of simultaneous B cell lymphomas (composite lymphoma) [64], [78], [79], [88], and [89]. These lymphomas are usually other low grade B cell neoplasm such as CLL/SLL or MZL. The reasons for this association are not clear but may represent a higher susceptibility of these patients for developing B-cell neoplasms. Some cases have been also found in patients with myelodysplastic syndrome or carcinoma, although this latter may just represent an incidental finding in the resected lymph node from patients with carcinoma [79] and [90].

7. In situ mantle cell lymphoma

Mantle cell lymphoma (MCL) is an aggressive mature B-cell lymphoma genetically characterized by the translocation t(11;14)(q13;q32) that juxtaposes CCND1 to IGH resulting in cyclin D1 overexpression, a cyclin that is not normally expressed in lymphoid cells. Genetic studies of the IGH and CCND1 breakpoints have demonstrated that also this translocation occurs mainly in pro-B/pre-B under influences of RAG endonuclease and AID [91] ( Fig. 2 ). Although this initial oncogenic event occurs in the bone marrow, the full neoplastic phenotype of this tumor is acquired at later stages of the B cell differentiation process. The predominant localization of the tumor cells in the mantle zone of the lymphoid follicle suggests that this microenvironment must play an important role in the pathogenesis of MCL. The recent identification of in situ MCL lesions would reinforce this idea. These lesions are characterized by the presence of cells that express cyclin D1 and carry the translocation t(11;14) restricted to a few layers of the mantle zone of follicles in the context of a reactive lymphoid tissues in otherwise healthy individuals [63], [92], and [93].

7.1. Multistep development of MCL

Similarly to the finding of circulating blood cells carrying the translocation t(14;18)(q32;q21), sensitive studies have also detected clonal populations with the t(11;14)(q13;q32) in the blood of 1–8% of healthy individuals [94] . These clones may persist for long time but its potential to develop an in situ or overt MCL is unknown [94] . The frequency of in situ MCL lesions in reactive lymphoid tissues seems extremely low since it has not been detected in any of the 100 and 131 reactive tissues investigated in two studies (<0.05%) [63] and [95]. The lack of detection of in situ MCL lesions contrasts with the 2–2.6% in situ FL observed in reactive lymphoid tissues [63], [64], and [74] and is concordant with the lower prevalence of clones carrying the t(11;14) compared to the t(14;18) in healthy individuals. Similarly to FL, the low frequencies of in situ and overt MCL in the general populations compared to the higher prevalence of clones with the t(11;14) in healthy individuals suggest that most of these circulating clones will never transformed into a malignant tumor. On the other hand, the report of the simultaneous expansion of two clonally identical MCL in the donor and recipient 12 years after allogeneic bone marrow transplantation highlights the long latency period needed by the initial clones to develop an overt MCL [96] . These clinical observations are concordant with the weak oncogenic potential of CCND1 to drive lymphomagenesis. Experiments in transgenic mice models have shown CCND1 requires the cooperation of stronger oncogenes such as MYC to develop lymphomas [97] . The secondary genetic abnormalities that may drive the progression from circulating clones to in situ lesions and overt MCL are unknown.

On the other hand, some in situ MCL will progress to overt lymphomas ( Fig. 2 ). Retrospective analysis in patients with MCL has identified in situ lesions in reactive lymphoid tissues obtained 2–15 years prior to the diagnosis of the lymphoma in most of the cases, suggesting that MCL proceeds through a stage of in situ lesions [98] . Interestingly, in situ MCL lesions have been also observed in an incidental lymph node of a patient in apparent complete remission of a previously diagnosed MCL, suggesting that the mantle zone of the lymphoid follicle may be an appropriate microenvironment to support chemoresistant MCL cells [63] .

7.2. Pathological findings of in situ MCL

Similarly to in situ FL, in situ MCL is usually an incidental finding in reactive lymphoid tissues ( Fig. 4 a–c). This diagnosis can only be made when cyclin D1 positive cells are detected by immunohistochemistry in the mantle zone of a reactive follicle. The cells are usually restricted to several layers predominantly in the inner area of the mantle zone, although in some cases may occupy the whole mantle. In these cases the mantle is not expanded and always intermingled with cyclin D1 negative cells ( Fig. 4 d). Edlefsen et al. [99] reported a rare case in which cyclin D1 positive cells were observed within the germinal center. In situ MCL cells usually express CD5 but some cases are negative [63] and [100]. SOX11 is a transcription factor highly specific of MCL that seems to play an important role in the aggressive behavior of this tumor [101] . Some MCL negative for SOX11 may correspond to a particular subtype of this lymphoma with a tendency to present with non-nodal leukemic disease and follow a more indolent clinical behavior [6] and [102]. Interestingly, most in situ MCL lesions express SOX11 but others are negative suggesting that the differential expression of this transcription factor occurs early in the development of MCL [63] and [87].

gr4

Fig. 4 Pathological findings of in situ MCL. HE stained section (a) and immunohistochemistry of CD20 (b) or CD10 (c) show a follicle with a germinal center. Significant abnormal architectures are not identified. Immunohistochemistry for cyclin D1 (d) highlights positive cell aggregation in inner mantle zones (Olympus BX51, magnification 12.5× for HE, 200× for CD10 and cyclin D1).

In situ MCL lesions have to be distinguished from overt MCL with a mantle zone growth pattern [63] and [85]. In the latter cases the architecture of the lymphoid node may show focal effacement with follicles involved, mantle zones expanded with images of back-to-back follicles and mantle zone entirely occupied by cyclin D1 positive cells. Focal extension to the interfollicular areas may be seen. The distinction between in situ MCL and MCL with mantle zone growth pattern is important because the latter is frequently associated with a disseminated disease [63] .

7.3. Clinical implications

The clinical information in individuals with in situ MCL lesions is still limited. No clear evidences of a simultaneous overt MCL have been reported in these patients [63], [93], and [103]. However, 4 of 6 patients with the diagnosis of a MCL with mantle zone growth pattern had disseminated disease at diagnosis [63] . The malignant potential of the in situ MCL lesions seems limited since only one of 15 patients, including 9 untreated, with more than one year follow-up, developed a disseminated MCL 4 years after diagnosis ( Table 2 ) [63], [93], and [103]. Five patients with in situ MCL lesions had also tumor cells in bone marrow and peripheral blood but none of them developed an overt lymphoma after 1–19.5 years follow-up, including three of them untreated [63] . These findings suggest that the circulating tumor cells in these cases may not have acquired a full malignant potential and therefore some caution should be used in the managements of these patients. Given the low malignant potential of these lesions, and similarly to the in situ FL lesions, the term “in situ involvement by MCL-like cells of uncertain significance” has been proposed [87] .

The potential value of SOX11 expression in predicting the evolution of in situ MCL lesions is still not known. The only reported patient that developed a disseminated disease was SOX11 positive and 5 of 6 cases with MCL with a mantle zone growth pattern were SOX11 positive. Five of 7 in situ MCL lesions in untreated patients with a long follow-up (1–19.5 years) without progression were SOX11-negative [63] .

Similarly to in situ FL, some in situ MCL lesions are associated with non-MCL lymphomas. These lymphomas are composed of low grade B cell lymphomas, such as FL, nodal MZL, extranodal MZL and CLL/SLL.

8. Conclusion

The wide application of flow cytometry, immunohistochemistry and molecular techniques has led to the incidental but frequent observation of monoclonal B-cell expansions in the peripheral blood and in lymphoid tissues of otherwise healthy subjects. The recognition of clonal expansions of cells carrying a neoplastic phenotype and/or oncogenic translocations (e.g. t(14,18) and t(11;14)) without fulfilling lymphoproliferative disorder criteria created a number of questions about their actual neoplastic nature. Although these clones may be considered conceptually as an early step in the development of CLL, FL and MCL, it is not yet clear why and how some cases progress to full-blown diseases while others remain stable.

B cell expansions with and without a CLL-like phenotype can be observed in the PB during routine blood examinations. Cell proliferations with features of CLL, FL or MCL but with a restricted distribution in follicles, germinal centers or mantle zones of the follicles may also be detected in clinical practice usually as incidental findings in the study of some lymphoadenopathies or other reactive lymphoid tissues. Similarly, the potential malignant progression of all these lesions is low or very low since most patients will never develop an overt leukemia/lymphoma, but it cannot be excluded in all cases. Thus, clinical monitoring have been recommended for high-count MBL, in situ FL and MCL although more studies are needed to define better the management guidelines for these individuals. In contrast, recent observations suggest that low-count MBL, where the clone size is below the threshold of diagnostic tests applied in clinical practice, do not need clinical follow-up as no progression has been demonstrated so far. The finding of such clones does not represent an actual clinical issue as they are mainly detected in the context of research studies.

Though the clinical value of all these entities remains uncertain, it is undeniable that their recognition has given great impulse to study mechanisms involved in lymphomagenesis and factors responsible for the transition from B-cell precursor states to overt lymphoproliferative disorders.

Conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgements

Authors are deeply grateful to Daniel Martinez, Paola Castillo, Itziar Salaverria and Cristina Royo, Claudia Fazi, Lorenza Pecciarini and Cristina Scielzo for providing pictures and fruitful discussions. This work was supported by grants from the Comisión Interministerial de Ciencia y Tecnología (CICYT) SAF08-03630 and SAF12-38432, and the Generalitat de Catalunya 2009SGR992 (E.C. is an ICREA-Academia researcher of the Generalitat de Catalunya); Associazione Italiana per la Ricerca sul Cancro AIRC (Investigator Grant to PG and Special Program Molecular Clinical Oncology – 5 per mille #9965), FIRB 2010 – Ministero Istruzione, Università e Ricerca (MIUR), Roma. K.K. has received a fellowship from the Uehara Memorial Foundation, Research fellowship (Japan).

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Footnotes

a Department of Anatomic Pathology, Hospital Clínic, Villarroel 170, 08036 Barcelona, Spain

b Laboratory of B-cell Neoplasia, Division of Molecular Oncology, San Raffaele Scientific Institute, Università Vita-Salute San Raffaele, Via Olgettina 58, 20132 Milano, Italy

c Clinical Unit of Lymphoid Malignancies, Department of Onco-Hematology, San Raffaele Scientific Institute, Università Vita-Salute San Raffaele, Via Olgettina 60, 20132 Milano, Italy

lowast Corresponding author. Tel.: +34 93 2275450; fax: +34 93 2275572.

1 KK and LS share the first authorship.

2 EC and PG share the senior authorship.