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Monoclonal B-cell lymphocytosis (MBL) is an asymptomatic haematological condition characterized by low absolute levels of B-cell clones with a surface immunophenotype similar to that of chronic lymphocytic leukaemia (CLL). In the general population, MBL increases with age with a prevalence of 5–9% in individuals over age 60 years. It has been reported to be higher among first-degree relatives from CLL families. We report results of multi-parameter flow cytometry among 505 first-degree relatives with no personal history of lymphoproliferative disease from 140 families having at least two cases of CLL. Seventeen percent of relatives had MBL. Age was the most important determinant where the probability for developing MBL by age 90 years was 61%. MBL clustered in certain families but clustering was independent of the number of known CLL cases in a family. As is the case with CLL, males had a significantly higher risk for MBL than did females (p=0.04). MBL patients had significantly higher mean absolute lymphocyte counts (2.4 × 109/l) and B-cell counts (0.53 × 109/l) than those with a normal B-cell immunophenotype. Our findings show that MBL occurs at a very high rate in high risk CLL families. Both the age and gender distribution of MBL are parallel to CLL, implying a shared inherited risk.
Monoclonal B-cell lymphocytosis (MBL) is an asymptomatic haematological condition characterized by small absolute levels of blood B-cell clone. MBL is characterized by a surface phenotype similar to that of chronic lymphocytic leukaemia (CLL) but minor subclasses are defined that closely resemble non-Hodgkin Lymphoma (Ghia, et al 2004). These clones are detectable at low cell numbers in otherwise healthy individuals using sensitive 4 – 8 color flow cytometric analysis. This technology was used to evaluate clonal B-cell expansion in adults (as a potential early marker of CLL) in a series of environmental health studies conducted around hazardous waste sites (Shim, et al 2007). Shanafelt et al. (2010) reviewed the prevalence of MBL from recent population studies and described the effects of the sensitivity of laboratory methods on resulting prevalences. Studies that have applied 4–6 colour flow cytometry have found MBL in 5–9% of the adult population over the age of 60 years with the prevalence increasing with advancing age (Dagklis, et al 2009, Ghia, et al 2004, Rawstron, et al 2002a). One study, using a more sensitive 8-colour flow and screening many more cells, found an MBL prevalence of 20% in this older age group (Nieto, et al 2009). A study using stored blood samples from individuals enrolled in a prospective population cohort who subsequently developed CLL showed that MBL could be detected virtually in all cases (45 of 46) up to 6 years before the diagnosis of CLL (Landgren, et al 2009). This study is strong evidence of MBL as a “preleukaemic” precursor state of CLL.
The International Familial CLL Consortium originally proposed diagnostic criteria for MBL(Marti, et al 2005). In addition to criteria for demonstrating clonal restriction with a typical CLL B-cell phenotype, the absolute number of B-lymphocytes (B-ALC) has to be less than 5.0 × 109 B-cells/l in the absence of any lymphoproliferative disease (LPD). Diagnostic criteria for CLL have recently been modified to specify a threshold of 5.0 × 109 B-cells/l [as opposed to an absolute lymphocyte count (ALC) of 5.0 × 109 /l] (Cheson, et al 1996, Hallek, et al 2008). This change reflects the fact that individuals with Rai stage 0 CLL with lower levels of absolute leukaemic B-cell counts are unlikely to progress and require treatment. As a consequence, individuals who were previously diagnosed with CLL in Rai stage 0 have a high likelihood of now being classified as MBL. Several studies have addressed factors that influence the prognosis of MBL(Fung, et al 2007, Rawstron, et al 2008, Shanafelt, et al 2009a) (Shanafelt, et al 2009b). For this type of analysis, one must make a distinction between MBL diagnosed in the clinic after referral for asymptomatic lymphocytosis (“clinical” MBL) and the detection of very small MBL clones in asymptomatic individuals from investigational screening studies (so called “low count” MBL), with the latter being far more prevalent in population-based studies (Dagklis, et al 2009, Nieto, et al 2009, Rawstron, et al 2002a). In one follow-up study, individuals with clinical MBL progressed to require CLL-specific treatment at a rate of about 1.1% per year. However, individuals with “low count” MBL (i.e. MBL detected in a healthy individual with a normal B-ALC (Dagklis, et al 2009)) have not been observed to progress over the course of several years (Fung, et al 2007, Rawstron, et al 2008), consistent with the hypothesis that the most important clinical determinant of progression to CLL is a B-cell count above the normal range (Shanafelt, et al 2009a).
CLL has a strong familial risk component, with first-degree relatives of patients showing an 8.5-fold increased risk compared to relatives of controls (Goldin, et al 2009). In flow cytometry studies, MBL has been reported in 13–18% of first-degree relatives of CLL patients in high-risk families (defined here as families with at least two confirmed cases of CLL)(Rawstron, et al 2002b),(Marti, et al 2003). This can be compared to 3–5% in the general population using laboratory methods with a similar detection sensitivity (Ghia, et al 2004, Rawstron, et al 2002a), suggesting that MBL is a marker of inherited predisposition to CLL. Matos et al. (2009) found that among 167 relatives of sporadic CLL cases, 4.1% (and 5/32 or 15.6% of those over age 60 years) had MBL. Combined with the earlier observation that virtually all CLL cases in the general population are preceded by MBL (Landgren, et al 2009), this suggests that there are shared genes, or as yet unidentified host factors, associated with both MBL and CLL that could be detected either in families or in the general population. The earlier family studies involved relatively small numbers of first degree relatives, with sample sizes of 33 (Marti, et al 2003) and 59 (Rawstron, et al 2002b). We now report the characteristics of MBL in the largest study to date, analysing 505 first-degree relatives of CLL patients in 140 high risk families that have been collected through our Genetic Epidemiology of CLL (GEC) consortium.
The GEC consortium is a collaboration of researchers from seven institutions (Mayo Clinic, M.D. Anderson Cancer Center, National Cancer Institute, University of Minnesota/Minneapolis Veteran Affairs (VA) Medical Center, University of California San Diego, Duke University/Durham VA Medical Center, and University of Utah) with the overall aim of investigating the genetic basis of CLL through the collection of high-risk CLL families (i.e., families with two or more relatives with confirmed CLL). A total of 505 first-degree relatives (without lymphoproliferative disease- LPD) from 140 high-risk CLL families had flow cytometry performed. Among the 140 families, 74 (53%), had 2 CLL cases reported, 41 (29%) had 3 CLL cases reported, and the remainder had 4 or more CLL cases. All participants provided written informed consent. A brief clinical history was obtained from all study participants. A complete blood count (CBC) was performed when possible, and was obtained in 329/505 (65%) of study participants. The protocol was approved by the Institutional Review Board at each centre. Nineteen of the 505 individuals were part of an earlier study (Marti, et al 2003).
Flow cytometry on fresh or previously frozen blood samples was conducted in laboratories at Duke University, the US Food and Drug Administration (FDA), and Mayo Clinic. The following MBL detection strategy was developed from previously published institutional methods (Lanasa, et al 2009, Landgren, et al 2009, Morice, et al 2008) and was employed at all sites. CBC was obtained using an automated blood cell counter and/or, in some cases, a single platform determination using BD TrucountTM (Becton Dickinson, San Jose CA) was made for the absolute lymphocyte subsets counts for fresh blood. Multi-parameter flow cytometric analysis was performed on either fresh (all sites) or cryopreserved peripheral blood mononuclear cells (PBMC; Duke and FDA). Frozen samples were cryopreserved in dimethyl sulfoxide containing media in aliquots of approximately 5.0 × 106 PBMC. Samples were rapidly thawed and washed twice with RPMI 1640 medium supplemented with 10% fetal bovine serum and 100U/ml penicllin/streptomycin. At least 2.5 × 105 PMBC were incubated with fluorescent conjugated CD5, CD19, CD20, κ , λ, and either CD45 or CD23. The fluorescent antibody-conjugates were selected by the lead flow-cytometry skilled investigator at each site (Table SI). VitalDye (Invitrogen, Carlsbad, CA, USA) was used for viability assessment in the analysis of cyropreserved samples. Flow cytometry was performed on a LSR II (Duke), FACSCalibur or FACSCantoII (FDA), FACSCanto or FACSCantoII (Mayo) instrument (Becton Dickinson).
Daily calibration of photomultiplier tube voltage settings were accomplished using microbead standards at all sites. Compensation controls were prepared by staining cells or microbead standards with a single flurochrome for software based corrections. The number of cell events collected varied from 1–5 × 105 cells. Listmode data were collected and analysed using FACSDiva (Becton Dickinson) or FloJo (Tree Star, Inc., Ashland, Oregon) software. A sample flow cytometry analysis is shown in Figure S1. The initial gating strategy used geometric gates to eliminate cellular aggregates and identified CD45-positive cells with typical lymphocyte forward and side scatter. CD19+ B-cells were identified and the surface expression of CD5 and CD20 was then determined to establish a B-cell phenotype. Three B cell subsets ( CD5−CD20+, CD5+CD20+, and CD5+CD20dim), were each analysed for skewing of the ratio of surface immunoglobulin κ to λ, and if needed for confirmation, expression of CD23. CLL-like MBL were defined as CD5+, CD19+, CD20dim ,sIgdim B cells that were CD23+ with a skewed κ : λ ratios (≥ 3:1 or <1:3). The definite formation of a cluster consisting of at least 30 cells or more was required for a positive MBL identification, yielding a detection sensitivity down to 1.0 × 10−4 cells or 0.01%. Atypical MBL were defined as CD5+ B cell populations with skewed κ : λ ratios (≥ 3:1 or <1:3) or having a separate, identifiable CD5+ B cell population that was distinct from the normal, polytypic B cells and not otherwise meeting criteria for CLL-like MBL. Non-CLL MBL were CD5− B cell populations with either skewed κ : λ ratios or having a distinct and separate CD5− B cell population.
To assure concordance among flow cytometry laboratories, several different strategies were employed. First, a series of 15 family members were screened for MBL at different laboratories. Among the 15 samples, all were concordant across the laboratories and included 2 with MBL and 13 with normal imunophenotypes. A comparison between ficoll-purified and whole blood samples was done between the FDA and Mayo Clinic and there was complete concordance among the nine samples (data not shown). Listmode data for all MBL samples were shared among the three flow cytometry lead investigators for confirmation of MBL detection. Finally, the three flow cytometry lead investigators held periodic conference calls to discuss cases with apparent small MBL clones so that a consensus judgement of MBL could be rendered.
Survival analyses were used to compute the hazard of a relative developing MBL using the lifetable method. Gender and number of CLL cases in the family were used as stratifying variables. Proportional hazards models were also applied in order to take into account the familial relationships among the study subjects. Non-parametric analyses were used to compare ALC and B-cell ALC values between MBL cases and individuals with normal immunophenotypes. To test whether MBL clustered in some families, we determined the expected number of MBL cases for families with 4 or more individuals tested using the age and gender specific probabilities from the lifetable model. This was compared to the observed number of cases using a chi-square statistic. All computations were performed using SAS version 9.1 (Cary, NC); p values < 0.05 were considered significant.
In our 140 families, 505 blood relatives without CLL or other LPD were analysed by flow cytometry. Forty three percent were male and the mean age at screening was 56.1 years. A total of 86/505 (17%) first-degree relatives were classified as MBL. 50 of the 86 MBL cases had ALC or B-ALC available; all 50 had <5.0 × 10 9/l B-cells and thus met MBL criteria. Among the 36 remaining MBL cases, we looked at the proportion of monoclonal B-cells and/or the proportion of B-cells to other cell populations (lymphocyte subsets) to exclude the possibility of CLL. This is because “clinical” MBL and early stage CLL are associated with a relative expansion of the leukaemic or clonal B cell compartment among the PBMC. “Low count” MBL individuals and individuals with a normal immunophenotype had 5 to 15% of PBMC comprised of CD19+ B cells. Only 2 of 36 MBL individuals without a CBC showed characteristics potentially suggestive of CLL: one individual showed that the B cell compartment constituted 42% of the PBMC. In the other MBL individual, B cells comprised 79% of the PBMC. Therefore, two of the 86 MBL cases identified among first-degree relatives identified in this study could potentially be early stage CLL rather than MBL, but this number is so low as to not affect the overall conclusions. In fact, if the overall prevalence of MBL among relatives with and without blood counts were compared, the results were not significantly different, being 15% (50/329) versus 20% (36/176), respectively.
Table I describes the sample characteristics and the number of MBL cases in various strata. As expected from population data, the prevalence of MBL increased with age in our sample. Consistent with population rates of CLL (Horner, et al 2009), males had an overall higher rate of MBL than females (19.5% vs. 15.5%). The survival curves by gender are shown in Figure 1. The overall probability for a relative to develop MBL by age 90 years was 61%. Males had a significantly higher risk of MBL than females (p-value=0.04) using a Cox proportional hazards model which takes into account the familial correlations among related subjects. The rate of MBL did not differ significantly by laboratory or by study site (Table I). If the sample was divided into relatives of families with the largest number of CLL patients (4 or more- a group which represented a third of the sample) and compared this to kindreds with 2 or 3 cases, the rate of MBL in these two groups did not differ significantly (results not shown). Thus, age was the most important factor associated with development of MBL in these families.
Because our families differed substantially in regards to both the size of family and the numbers of individuals screened for MBL, we also examined the deviation of observed vs. expected numbers of MBL by family. Among the 140 families tested, 46 families had at least 4 individuals tested by flow cytometry, and 7 of these 46 (15%) families had significantly more MBL than expected by chance. No family had fewer MBL cases than expected but there was limited power to detect this type of decrease because the diagnosis rate of MBL is low before age 70 years.
An ALC was available for 65% of the individuals in our sample. Comparisons between MBL and normal immunophenotype individuals are shown in Table II and in Figures 2 and and33 for ALC and B-ALC values, respectively. The MBL cases had a significantly higher ALC than those with normal immunophenotypes (Figure 2). Two MBL individuals had ALC values above the normal range but in neither of the cases did the B-ALC meet the numeric criteria for CLL. Although each laboratory had slightly different definitions of lymphocytosis, Shanafelt and Hanson (2009) recently suggested that a cut-off of ALC > 3.0 × 109/l be applied to classify MBL as “clinical” (e., most likely to be detected through routine CBC testing). Using this definition of absolute lymphocytosis, 18% (9/50) of the MBL cases had “clinical” MBL. Among individuals with normal immunophenotype, only 10% had an ALC > 3.0 × 109/l. If a more stringent criterion for lymphocytosis (ALC =4.0 × 109/l or more) was used, 1.8 % of those with normal immunophenotype but 3/50 (6%) of MBL cases had lymphocytosis, respectively. B-ALC values were available for some individuals (Figure 3). Again, MBL cases had significantly higher B-ALC than those with a normal immunophenotype. As shown in Figures 2 and and3,3, the distributions of ALC and B-ALC were both shifted higher in MBL patients compared to normals and differed significantly using non-parametric tests. In analysing the type of MBL found in our sample, most were CD5+CD20 dim (CLL-like). However, 12 cases were not CLL-like; 7 of these were atypical (i.e. CD20 bright) and 5 cases were CD5-. Among MBL cases, the percentage of B-cells that exhibited the CLL phenotype averaged 35% with a range of 1–95%. Thus, MBL cases identified from these families were mostly low ALC.
This study used flow cytometry to systematically screen the largest number of first-degree relatives of CLL cases from high-risk families reported to date. The overall rate of 17% MBL is consistent with that noted previously in smaller studies of familial CLL(Marti, et al 2003, Rawstron, et al 2002b). Our results strongly suggest that MBL in these families represents an inherited predisposition to CLL and that CLL and MBL share genetic risk factors. In the general population, the incidence rates for CLL are very low before the age of 40 years and then increases progressively after age 50 years with the rates in males being about twice that in females for every age category(Horner, et al 2009). We found that the increase of MBL with age showed a similar pattern to that of CLL. In our high-risk families, the probability of having MBL increases with age and is 61% by 90 years of age. In another parallel with CLL, the rate of MBL was significantly higher in males than females although the difference was smaller than in CLL. Other population studies of MBL have not shown consistent gender differences(Dagklis, et al 2009, Ghia, et al 2004, Nieto, et al 2009, Rawstron, et al 2008). It is possible that gender differences are more important for progression to clinical disease than for initiation of CLL-like changes.
In evaluating this body of work it is important to consider the technical aspects of the flow assays utilized when comparing rates of MBL detection across studies. Our flow cytometry methodology is similar to the detection sensitivity used in previous population-based screenings that found overall rates of 3.5%–7.4%, which are much lower than the overall rate of 17% observed in our high-risk family members. In a recent large study of an Italian population isolate, Dagklis et al. (2009) reported an overall rate of MBL of 7.4% with more than 14% in individuals over 70 years old. Matos et al. (2009) found that 15.6% of first-degree relatives of sporadic CLL cases above the age of 60 years had MBL. In our high risk families, 48/400 (24%) of relatives over 60 years of age and 28% of relatives aged over 70 years had MBL (see Table I). Representing an outlier is the report by Nieto et al (Nieto, et al 2009), in which MBL prevalence was 12% in a population-based cohort. This study used a multi-parameter flow cytometry technique with a detection sensitivity approximately 10-fold higher than that of prior studies and the current study, which makes meaningful direct comparison difficult. Overall, these results all suggest that MBL reflects a genetic association to CLL in which risk increases in a stepwise manner, least in the general population, higher in relatives of sporadic CLL, and highest for relatives of familial CLL.
More than 85% of MBL cases had a CLL-like immunophenotype (CD5+, CD19+, CD20 dim, CD23+, sIg dim) in our study. The percentages were lower in other population studies, (Ghia, et al 2004, Rawstron, et al 2008). There may be heterogeneity among these families in terms of whether the type of MBL (i.e. CLL-like, atypical, or CD5−) clusters as a familial characteristic although we did not see evidence of familial clustering. While we did identify a few families with significantly more MBL cases than expected by chance, the rarity of MBL at younger ages and the uneven distribution of numbers of relatives available for testing in each family made it difficult to conclude that any particular family had less MBL than expected. Familial clustering of MBL was also reported in the population isolate studied by Dagklis et al.(2009), where 28% of the MBL cases were clustered in 5 extended families.
In terms of clinical significance, most of the relatives with MBL that we identified had normal ALC and B-ALC and thus are at very low risk for progression to CLL or other LPD. However, more longitudinal data are needed to determine if the clinical outcomes of low cell count MBL identified in CLL families are the same as in the general population. Further, longitudinal evaluation of this cohort will clarify whether relatives with MBL are at greater risk for developing CLL or another LPD malignancy than are relatives without MBL.
Our prior studies have shown a strong familial aggregation of CLL and other indolent B-cell lymphomas (Goldin, et al 2009). Our current results, documented in this study, that MBL is also part of the “CLL spectrum” of conditions that share common genes. If MBL is an early step in the process of CLL development, then germ line genes are likely to be acting early in leukaemogenesis, contributing to initial oncogenic events required before frank CLL development is manifest. Conducting global genetic studies on both MBL and CLL patients will probably help identification of the genes and pathways involved in both the initiation and progression of CLL.
This material is based upon work supported by the Intramural Program of the National Cancer Institute, National Institutes of Health, Bethesda, Maryland, and by grants CA118444 and CA116237 from the National Cancer Institute. JBW is also supported in part by grant CA137941 from the National Cancer Institute, grant # 61006 from the Leukemia and Lymphoma Society, and the Veterans Affairs Research Service. MCL is a Fellow of the Leukemia and Lymphoma Society of America. Flow cytometry was performed in the Duke Human Vaccine Institute Flow Cytometry Core Facility that is supported by the National Institutes of Health award AI-51445. Data collection in Utah was made possible by the Utah Population DataBase (UPDB) and the Utah Cancer Registry (UCR). Partial support for all data in the UPDB was provided by the University of Utah Huntsman Cancer Institute. The UCR is funded by contract N01-PC-35141 from the NCI’s SEER program with additional support from the Utah State Department of Health and the University of Utah.
Conflict of interest disclosure: The authors declare no competing financial interests.