(2010) 24, 512–520 & 2010 Macmillan Publishers Limited All rights reserved 0887-6924/10 $32.00 www.nature.com/leu REVIEW

Monoclonal B-cell (MBL): biology, natural history and clinical management

TD Shanafelt1, P Ghia2, MC Lanasa3, O Landgren4 and AC Rawstron5

1Division of , Department of Medicine, Mayo Clinic, Rochester, MN, USA; 2Laboratory of B Cell Neoplasia, Division of Molecular and Unit, Department of Oncology, Universita Vita-Salute San Raffaele and Istituto Scientifico San Raffaele, Milano, Italy; 3Department of Medicine, Duke University Medical Center, Durham, NC, USA; 4Medical Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA and 5Hematological Malignancy Diagnostic Service, St James0s Institute of Oncology, Leeds Teaching Hospitals, Leeds, UK

Chronic lymphocytic leukemia (CLL) and the other low-grade phenotype, (ii) atypical-CLL phenotype or (iii) non-CLL pheno- non-Hodgkin are among the most common lym- type based on surface protein expression (Table 1).6 phoid malignancies. Recent studies suggest that more than 4% of the general population over age 40 harbor a population of As the prevalence of MBL is several hundred times more clonal B cells with the phenotype of either CLL or another B-cell common than CLL or the NHLs of corresponding phenotype, the malignancy, a condition now designated monoclonal B-cell above studies raised the possibility that CLL and the low-grade lymphocytosis (MBL). Although all cases of CLL appear to be NHLs may have highly prevalent precursor states analogous to preceded by MBL, the majority of individuals with MBL will not the relationship between of undeter- develop a hematologic malignancy. The biologic characteristics mined significance and or adenomatous and clinical implications of MBL appear to differ based on whether it is identified during the diagnostic evaluation of polyps and colon cancer. This discovery has raised a host of lymphocytosis or incidentally discovered through screening of clinical and biologic questions that have been the focus of individuals with normal counts as part of research intense investigation over the last decade. In this paper, we studies using highly sensitive detection methods. In this paper, provide a state of the art review on the prevalence, biology, we provide a state of the art review on the prevalence, natural history, nomenclature/classification and clinical nomenclature, biology, natural history and clinical manage- management of MBL. ment of MBL. Leukemia (2010) 24, 512–520; doi:10.1038/leu.2009.287; published online 21 January 2010 Population prevalence of MBL Keywords: monoclonal B-cell lymphocytosis (MBL); chronic lymphocytic leukemia (CLL); prognosis; biology; management; Several studies have investigated the prevalence of MBL in the non- general population. The proportion of individuals with MBL and the absolute numbers of abnormal cells detected in these studies Introduction are closely associated with the methods used for detection. A summary of published studies is shown in Table 2. The earliest population studies were performed in the United Chronic lymphocytic leukemia (CLL) is the most common States in the 1990s as part of a larger investigation into the leukemia in the Western world and accounts for approximately 2 1 potential health effects of living around hazardous waste sites. 11% of all hematologic malignancies. CLL affects approxi- These studies included an immune biomarker panel with basic mately 3–5 out of every 100 000 individuals in the United States lymphocyte immunophenotyping but not B-cell kappa:lambda and is presently considered a treatable but incurable illness with analysis. As discussed in the introduction, the overall prevalence conventional therapies. Remarkably, while conducting a public of MBL from the initial study and two follow-up analyses using health study in 1991, the US Center for Disease Control this approach was 9/1499 (0.6%) individuals.3 Later studies identified a clonal population of B cells with CLL phenotype performed independently in the United Kingdom and Italy in 9/1499 (0.6%) individuals in the general population age 45 2,3 assessed the prevalence of clonal B cells in individuals with or older. This prevalence of roughly 1 out of every 170 normal counts using four-color flow cytometry with a individuals was several hundred times more common than that sensitivity of detection commonly used for detection of minimal of CLL. Subsequent studies found that clonal B-cell populations residual disease in patients with CLL (1 clonal cell per 1 Â 105 with the phenotype of other low grade, non-Hodgkin lymphoma events).7 These studies showed a much higher prevalence of (NHL) subtypes (for example, marginal zone) were also 4,5 CLL-like MBL, which was detected in more than 5% of adults prevalent in the general population. To standardize char- aged over 60.4,5 The UK study4 involved hospital outpatients acterization and promote future scientific investigation, an with no history or suspicion of cancer and the Italian study5 international panel of experts designated the presence of such involved individuals from a rural community referred for routine clones ‘monoclonal B-cell lymphocytosis’ (MBL) and suggested 6 blood tests (for example, blood glucose, blood lipids). CLL- formal criteria for the diagnosis of MBL in 2005. These criteria phenotype cells in these patients typically represented 10% of also sub-classified individuals with MBL as having (i) CLL-like o total B cells and absolute CLL-like cell counts were usually below 15 CLL-like cells/ml. In both studies, the prevalence Correspondence: Dr TD Shanafelt, Division of Hematology, Depart- increased with age and a higher proportion of men were ment of Medicine, Mayo Clinic, Rochester, MN USA. 4,5 E-mail: [email protected] affected. In addition to CLL-like clones, non-CLL-phenotype À Received 30 September 2009; accepted 15 October 2009; published MBL (CD5 ) cases were detected in 1–2% of individuals based online 21 January 2010 on a perturbation of kappa:lambda ratio. Monoclonal B-cell lymphocytosis TD Shanafelt et al 513 Both the United Kingdom and Italian groups have subse- flow cytometry approach and identified a higher prevalence quently performed further studies of MBL in the general (7.4%) of MBL in the general population than in the primary population with a focus on biological investigations.4,5 It is care cohort.8 An even more recent study from Salamanca used noteworthy that the second Italian study used a higher sensitivity the highest sensitivity flow cytometry approaches available to analyze 5 million peripheral blood cells per individual. Using Table 1 Diagnostic criteria and sub-classification for MBL (adapted this strategy the investigators identified a very high prevalence of 6 from Marti et al. ) CLL phenotype cells in the general population, which were detectable in more than one in five individuals over 60 years Diagnostic criteria old.9 As indicated by the latter two studies, the differences in a (1) Documentation of clonal B-cell population by one or more of reported prevalence of MBL across series are most likely due to following: differences in the sensitivity of the flow cytometry approach (a) Light chain restriction: Overall kappa:lamda ratio 43:1 or o0.3:1 or applied. In particular, the Salamanca group screened at least 425% of B cells lacking or expressing low-level surface 10 times more cells (5 million per case vs 200 000–500 000) Immunoglobulin and used eight-color staining panels as compared with the previously used four-color5 and five-color8 protocols. The (b) Heavy chain monoclonal IGHV rearrangements percentage of aberrant/clonal B cells was below the maximum (2) Presence of a disease-specific immunophenotypeb 9 sensitivity of the previous studies (o0.01%) in more than half of (3) Absolute B-cell count o5 Â 10 cells/l 9 (4) No other features of a lymphoproliferative disorder or the cases (62%) identified by the Salamanca study. However, it autoimmune disease should be noted that clonality was only confirmed by additional (a) Normal physical exam (no or organomegaly) methods in 18 cases, all except one with 40.01% aberrant/ (b) Absence of B-symptoms (for example, fatigue, weight loss clonal B cells.9 and night sweats) attributable to NHL The highest reported prevalence of CLL-like MBL occurs (c) No autoimmune/infectious disease in first-degree relatives of CLL patients. All epidemiological Subclassification studies that have investigated the prevalence of CLL and other (A) CLL-like phenotype lymphoproliferative disorders in relatives of CLL patients have Co-expression of CD5 with CD19; CD20 (dim); and CD23 reported elevated risks of CLL in relatives with the largest study Light chain restriction with dim surface immunoglobulin showing a eightfold increase in risk.10,11 The first reports of MBL expression (very small MBL clones may be oligoclonal came from studies of apparently unaffected family members in and thus not light chain restricted) the early 1990s.12 More recently studies in both the United 13 12 (B) Atypical-CLL phenotype Kingdom and United States showed a very high prevalence Co-expression of CD5 with CD19 but CD23 negative or CD20 of CLL-like MBL in individuals with a family history of CLL who (bright) had normal blood counts (prevalence ¼ 13.5–18%). The UK Light chain restriction with moderate-to-bright surface group had used the same methodology for their studies of both immunoglobulin expression CLL families and the general population and found a fourfold Exclude t(11;14) to rule out increase prevalence of MBL in families with a genetic pre- (C) Non-CLL phenotype disposition to CLL.4,13 For young adults aged 16–40 years the CD5 negative relative risk is 17-fold.13 Although these studies12,13 clearly show Express CD20 a higher risk of MBL among unaffected individuals from CLL Light chain restriction with moderate-to-bright surface families relative to the general population, other recent studies immunoglobulin expression suggest that first-degree relatives of individuals with sporadic CLL Abbreviations: CLL, chronic lymphocytic leukemia; IGHV, immunoglo- may also be at increased risk of MBL if they are over age 60.14 bulin heavy chain variable; MBL, monoclonal B-cell lymphocytosis; As noted, the sensitivity of the flow cytometry approach used NHL, non-Hodgkin lymphoma. aWhere possible, repeat assessment should show the monoclonal for detection has a direct effect on the absolute numbers of CLL- B-cell population is stable over 3-month period. phenotype cells that can be identified and hence the proportion bIn the absence of a disease-specific immunophenotype, a highly of individuals with detectable CLL-like MBL. The initially skewed kappa:lamda can be the result of a reactive process. suggested diagnostic criteria for MBL shown in Table 1 did not

Table 2 Prevalence of CLL-like MBL in population studies

Study group CLL-like MBL prevalence

Source Median age N No. of CD19/CD5 + k/l CD19/CD5 + CD20 Events All ages 460 years (range) colors ( Â 103) in study

US residential population3 53 (40–78) 1926 2 No No NS 0.6% 40.6%a US blood donors41 45 (18–79) 5141 2 No No NS 0.14% 0.9% UK hospital outpatients18 57 (40–90) 910 4 Yes Yes 200 3.5% 5.0% Italy primary care5 74 (65–98) 500 4 Yes Yes 200 5.5% 5.5%b UK hospital outpatients18 74 (60–80) 1520 4 No No 200 5.1% 5.1%c Italy residential population8 55 (18–102) 1725 5 Yes Yes 500 7.4% 8.9% Spain primary care9 62 (40–97) 608 8 Yes Yes 5000 12.0% 420%a Abbreviations: CLL, chronic lymphocytic leukemia; MBL, monoclonal B-cell lymphocytosis; NS, not specified. aEstimated from data. bAge above 65. cAge range 60–80. The detected prevalence is highly dependent on the method of detection.

Leukemia Monoclonal B-cell lymphocytosis TD Shanafelt et al 514 specify a flow cytometry analysis method, and were simply Although these findings suggest a significant biologic overlap based on detection of a monoclonal B-cell population in the between CLL and CLL-like MBL, it is important to emphasize peripheral blood with an overall kappa:lambda ratio 43:1 or that all these studies were performed on MBL cases with a high o0.3:1, or 425% of B cells lacking or expressing low level number of circulating monoclonal cells to facilitate these surface immunoglobulin (Ig) in conjunction with a disease- analyses. In routine clinical practice, a vast majority (B85%) specific immunophenotype.6 These criteria are primarily in- of MBL cases are identified after investigation of lymphocytosis tended for diagnostic laboratories undertaking evaluation of and have an abnormal B-cell count above 1900 cell/ml. In samples referred for investigation of a suspected lymphoproli- contrast, B85% of MBL detected on population-screening ferative disorder. The ability to detect very small abnormal studies have a B-cell count below 500/ml, with 40% having B-cell populations representing o100 cells/ml is generally not fewer than 50 CLL phenotype cells/ml. As several studies suggest required and high-sensitivity approaches are not needed for that the number of circulating B cells is a strong predictor of the routine diagnostic purposes. clinical outcome of MBL patients,18–20 it is reasonable to It should be noted, however, that it is important to postulate that, from a biological point of view, MBL detected in discriminate between CLL-like MBL, which has CD5 and clinical practice (‘clinical MBL’) may be more similar to frank CD23 expression along with weak CD20, CD79b and surface CLL than MBL detected on population screening (‘population- Ig, and other subtypes of MBL. The two most common non-CLL screening MBL’). types are CD5– MBL (classified as ‘non-CLL-phenotype MBL’) In spite of the similarities between CLL and clinical MBL and CD5 þ MBL with a phenotype that is not typical for CLL, reviewed above, several biologic differences between clinical that is, strong CD20, CD79b or Ig expression (classified as MBL and population-screening MBL have recently been ‘atypical-CLL-phenotype MBL’). Individuals with a CD5 þ , identified. First, although MBL was originally thought to CD23 negative phenotype must have the diagnosis of mantle represent exclusively monoclonal , it has cell lymphoma excluded by fluorescence in situ hybridization become evident that biclonal9, oligoclonal15 and polyclonal8 (FISH) evaluation for the t(11;14) before being classified as cases of MBL can be identified. This observation indicates that a having atypical-CLL-phenotype MBL. CLL-like phenotype (CD5 þ , CD20dim) is not necessarily related The optimal flow cytometry analysis approach for biologic to the acquisition of monoclonality. These studies suggest the studies aimed at investigating the earliest stages of leukemogen- possibility that concomitant clones of CLL-like B lymphocytes esis will depend on the numbers of abnormal cells required for appear and persist in many otherwise healthy individuals validation and the planned downstream analysis. Studies in which one clone may expand and, in a small subset of investigating CLL-like MBL designed to detect at least 1 CLL individuals, become predominant with time. Further investiga- cell/ml will require analysis of at least 200 000 total events using tion is now needed to elucidate the type of stimulation, antigen a minimum of five-color analysis (CD5, CD19, CD20, kappa activation or other events that induce some B lymphocytes to and lambda). In many cases, the detection of an abnormal acquire the CLL phenotype. population can be confirmed by consensus immunoglobulin Although the studies are far from conclusive, differences in Ig heavy chain (IGH)–PCR on separated B cells. Analysis of CLL repertoire have also been observed between individuals with cells below this level will typically require analysis of more than clinical MBL and those with population-screening MBL. CLL is 500 000 total cells with six or more fluorescent channels (CD3, characterized by a biased usage of immunoglobulin heavy CD5, CD19, CD20, kappa and lamda). Such studies need to be chain variable region (IGHV) genes with IGHV1–69, IGHV4–34, carefully controlled and validation of the abnormal population IGHV3–07 and IGHV3–23 the most frequently used. The requires fluorescent cell sorting of the abnormal population presence or absence of somatic mutations in the IGHV genes coupled with FISH analysis and IGHV gene sequencing after in patients with CLL correlates with clinical outcome in which cloning or extraction of DNA from single-sorted cells.15 individuals with mutated IGHV genes having a longer time to first treatment and improved overall survival (OS).21,22 Approxi- mately 55% (range 47–58%) of CLL patients have mutated IGHV Biology and nomenclature genes, although this frequency largely depends on whether individuals present þ primary vs tertiary referral center. The CLL-like MBL has been so-named because of its close IGHV1–69 gene is the most frequently used gene in unmutated phenotypic resemblance with clinical CLL. Shared markers CLL cases, while the IGHV4–34 gene is the most commonly used used in the diagnostic work-up include, beside CD5 and CD20, gene among patients with mutated CLL.23 Patients with clinical also CD23, low levels of surface Igs and Ig-related molecules MBL appear to be predominantly IGHV mutated (77–90%)18,19 (CD79b), and low-to-negative expression of FMC7.4,5 Further and have an IGHV repertoire that closely resembles that of analyses have been performed to define specific protein mutated CLL with IGHV3–07, IGHV3–23 and IGHV4–34 genes expression that may distinguish MBL from CLL. One study used in around half of the cases.18 Although some of these evaluated 18 additional B-cell markers by flow cytometry in genes (that is, IGHV3–07, IGHV3–23 and IGHV4–34) have been individuals with CLL, MBL, other B-cell lymphoproliferative also found to be expressed in familial population-screening disorders and individuals without B-cell malignancy.16 This MBL,15 the overall IGHV repertoire of population-screening analysis confirmed the distinct phenotype of CLL compared with MBL appears strikingly different.8,9 Specifically, the absence of other B-cell disorders, but found individuals with CLL and IGHV1–69 and an under-representation of both IGHV4–34 and CLL-like MBL clustered together when an unsupervised analysis IGHV3–23 genes was apparent in individuals with population- was performed. Expression of no specific protein was able to screening MBL relative to those with CLL or clinical MBL.8,9 distinguish between CLL and CLL-like MBL. Although a biased usage of the IGHV4–59/61 gene in popula- A similar conclusion was reached using gene expression tion-screening MBL has been suggested,8 larger data sets are profiling analysis. Although a single gene; lymphoid enhancer needed to determine whether or not a distinct restriction in IGHV binding factor 1 (LEF1), was capable of distinguishing CLL cells repertoire occurs in patients with population-screening MBL and from controls and all other B-cell disorders evaluated, the to define the random origin from the unselected repertoire expression of LEF1 in CLL and CLL-like MBL was similar.17 expressed by the normal B lymphocyte pool.

Leukemia Monoclonal B-cell lymphocytosis TD Shanafelt et al 515 Finally, 20–25% of CLL cases are characterized by the as detected by FISH were observed between individuals with existence of stereotyped heavy chain complementarity deter- MBL and Rai stage 0 CLL with an ALC p10 Â 109/l. The mining region 3 sequences within the functional heavy chain percentage of MBL patients free of treatment at 1, 2 and 5 years rearrangement, which suggests the recognition of repetitive and was 99, 98 and 93%, respectively.19 Among MBL patients, shared antigenic epitopes in unrelated patients with CLL.24 In B-cell count as a continuous variable (hazard ratio ¼ 2.9, P ¼ 0.04) contrast, stereotyped heavy chain complementarity determining was predictive of time to treatment.19 It is noteworthy that no region 3 sequences are rarely present (o5%) among individuals difference in survival was observed based on the percentage of with population-screening MBL, underscoring another biologic B cells that had CLL-like phenotype among those individuals difference with frank CLL.8 whose total B-cell count was o1.5 Â 109/l (P ¼ 0.71) suggesting that having a high proportion of clonal B cells does not necessarily indicate an increased risk for progression among Natural history and risk of progression individuals with a total B-cell count o1.5 Â 109/l. Among other demographic and prognostic parameters, CD38 status (hazard From MBL to CLL ratio ¼ 10.8; P ¼ 0.006) was associated with time to treatment, A major clinical concern for persons diagnosed with MBL is but age (P ¼ 0.62) and sex (P ¼ 0.99) were not.19 As a result of an whether or not they will develop CLL or another indolent NHL. insufficient number of patients with ZAP-70, IGHV gene Risk factors for progression are of key interest. At this time, only mutation status, or FISH analysis, the investigators were unable a few studies have addressed these questions. to correlate these variables with time to treatment or OS. Taken In a prospective hospital-based cohort study from the United together, the rate of progression from MBL to CLL requiring Kingdom, Rawston et al.18 monitored 185 subjects with CLL- treatment during the first 5 years of follow-up was approximately phenotype MBL and lymphocytosis for a median of 6.7 years 1.4% per year in this retrospective study of 302 patients. Primary (range 0.2–11.8 years) and found progressive lymphocytosis in 51 indications for treatment for the MBL patients in this series were (28%). Of these, 28 (15% of the entire cohort) progressed to a progressive lymphadenopathy (n ¼ 2), marrow failure/cytopenia diagnosis CLL. In this study, the absolute B-cell count was the (n ¼ 3), progressive lymphocytosis (n ¼ 1) and autoimmune only independent prognostic factor associated with progressive hemolytic anemia (n ¼ 1).19 lymphocytosis.18 Only 13 (7%) individuals in this cohort required In a separate prospective, hospital-based cohort study from chemotherapy for treatment of CLL. During the follow-up period, Italy, similar in design to the Mayo Clinic study,19 Rossi et al.27 62 (34%) subjects died, however, only 4 of these deaths were due compared the outcome of patients with MBL with individuals to CLL. Age at enrollment 468 years and hemoglobin level with Rai stage 0 CLL. In a consecutive series of 460 newly below 12.5 g per 100 ml were the only independent prognostic diagnosed cases with B-cell lymphocytosis of CLL phenotype, factors for death.18 Thus, based on 187 cases with a median the investigators used the International Workshop on Chronic follow-up of 6.7 years, this study reported that CLL requiring Lymphocytic Leukemia (IWCLL) 2008 guidelines26 to classify treatment develops in subjects with CLL-phenotype MBL and individuals as having either MBL or CLL (B-cell count o and lymphocytosis at the rate of 1.1% per year.18 X5 Â 109/l, respectively). The investigators identified 123 MBL In a retrospective laboratory-based database study, Mulligan cases and 337 CLL cases (Rai 0: 154; I–II: 129; III–IV: 54). et al.25 identified 414 MBL patients, 322 of whom had a CLL When compared with the 154 individuals with Rai 0 CLL, the phenotype. On the basis of 220 patients with adequate 123 individuals with MBL had a lower percentage of bone longitudinal information (median follow-up 4.1 years, range marrow lymphocytes (median 20 vs 40%, Po0.001), a lower 1.0–8.5 years), they found 76 (34.5%) patients progressed to prevalence of diffuse bone marrow involvement (2.4 vs 10.2%, CLL26 including 28 (12.7%) patients who developed an absolute P ¼ 0.034), and lower beta-2-microglobulin (median 1.9 vs lymphocyte count (ALC) of more than 20 Â 109/l and 18 (8.2%) 2.2 mg/l, P ¼ 0.002).27 MBL cases were also found to have more an ALC of more than 30 Â 109/l, the criterion for progression preserved immune function than those with Rai 0 CLL. For used by Rawstron et al.18 (that is, a rate of progression of example, MBL cases had higher Ig levels: IgG (median 10.60 vs approximately 2% per year). Further, they identified five MBL 9.74 g/l, P ¼ 0.012), IgA (median 1.55 vs 1.40 g/l, P ¼ 0.038) and cases with an initial B-cell count of o1.9 Â 109/l who IgM (median 0.74 vs 0.58 g/l, P ¼ 0.003) than those with Rai 0 progressed to fulfill the current criteria for CLL: three cases CLL cases. Clinically, a lower risk was observed in progressed to have an ALC that only modestly exceeded the individuals with MBL compared with those with Rai 0 CLL.27 current CLL diagnostic criteria,26 and two cases had progressive Before initial chemotherapy treatment, the incidence of infec- lymphocytosis to 26.8 Â 109/l and 52.5 Â 109/l after 3.1 and 2.8 tion was 10.9 vs 15.1 per 100 patient-years (P ¼ 0.030), for those years, respectively. with MBL and CLL respectively.27 In a retrospective clinic-based cohort study from the Mayo Rossi et al.27 also found a lower prevalence of high-risk Clinic, Shanafelt et al.19 examined the outcome of patients with cytogenetic lesions in individuals with MBL compared with CLL-like MBL relative to that of individuals with Rai stage 0 CLL. those with Rai 0 CLL. Specifically, MBL cases had a lower Using hematopathology records, the investigators were able to prevalence of del(11q22)/del(17p13) (3.8 vs 15.2%, P ¼ 0.004) identify 631 patients with newly diagnosed MBL or Rai stage 0 and of TP53 mutations (3.0 vs 11.5%, P ¼ 0.049) compared with CLL. Within this cohort, 302 patients fulfilled the criteria for those with Rai 0 CLL.27 In contrast, the percentage and diagnosis of MBL (B-cell counts of 0.02 to 4.99 Â 109/l); and 313 prevalence of IGHV gene identity; distribution of IGHV genes; Rai stage 0 CLL (94 patients had Rai stage 0 CLL with an ALC and prevalence of stereotyped heavy chain complementarity p10 Â 109/l; 219 patients had Rai stage 0 CLL with an ALC determining region 3 did not differ significantly between MBL 410 Â 109/l). Clinical information regarding date of diagnosis, and Rai 0 CLL cases.27 The expression patterns of CD38, ZAP-70 physical examination, prognostic parameters, treatment history and CD49d were not statistically different between the two and disease-related complications were abstracted from clinical groups.27 records. In the Italian study, 56 of the 123 MBL cases progressed to In this cohort, no differences in CD38 status, IGHV gene fulfill criteria for overt CLL (n ¼ 37) or small lymphocytic mutation status, ZAP-70 expression or cytogenetic abnormalities lymphoma (SLL) (n ¼ 19). The median time to developing CLL/

Leukemia Monoclonal B-cell lymphocytosis TD Shanafelt et al 516 SLL was 55.0 months. Intuitively, and consistent with the report Distinguishing MBL from CLL in clinical practice by Rawstron et al.,18 MBL cases with higher B-cell counts at MBL diagnosis were more likely to subsequently develop a As the clonal B cells of individuals with both CLL and CLL-like B-cell count above the 5 Â 109/l threshold currently used MBL share an identical immunophenotype, how to best to make the diagnosis of CLL.27 Overall, 19 of the 123 MBL differentiate MBL and Rai stage 0 CLL continues to be an area cases progressed to symptomatic CLL/SLL requiring treatment, of controversy. From a historic perspective, the 198829 and accounting for a 10-year treatment-free survival (TFS) of 68.7%. 199630 diagnostic criteria for CLL classified individuals with a In univariate analysis, IGHV gene mutation status (Po0.002), clonal population of characteristic immunophenotype and an CD38 expression (Po0.001), CD49d expression (P ¼ 0.006) ALC 45 Â 109/l as having CLL. After recognition of MBL and and cytogenetic abnormalities on FISH testing (Po0.001) all publication of the 2005 MBL diagnostic criteria,6 which were predicted treatment-free survival among individuals with MBL. based on B-cell count rather than ALC, there was initially On multivariable analysis, the presence of þ 12 or del(17p13) overlap between the diagnostic criteria for CLL and MBL: on FISH karyotype were the only independent predictors of TFS individuals with an ALCX5 Â 109/l who had a B cell o5 Â 109/l (hazard ratio ¼ 5.39, P ¼ 0.001).27 fulfilled both the MBL and CLL diagnostic criteria. Although this There remains great need for large, prospective MBL studies initially appeared to effect a small proportion of patients, to better understand the natural history of MBL and define subsequent studies indicated up to 40% of individuals with predictors of progression. On the basis of the similarities newly diagnosed Rai stage 0 CLL according to the 1988 and between three18,19,25 of the four large series,18,19,25,27 our 1996 criteria fell in this area of overlap.20,31 current estimate is that individuals with MBL have a 1–2% The distinction between classifying a patient as having annual risk of requiring CLL-specific treatment. Future studies leukemia, as opposed to a pre-malignant condition, should be may identify the biologic characteristics associated with the risk based, at least in part, on the individual’s risk of developing of progression, which in turn may allow physicians to better clinical complications and/or dying from the disease.20,31 In this predict the risk of progression for a given individual. It is regard, studies have now shown that the ALC threshold used in imperative that future prognostic studies in MBL focus not on the the 1988 and 1996 CLL diagnostic criteria has no relationship to likelihood of the B-cell count rising above an arbitrary threshold either TFS or OS while the B-cell threshold suggested in the (for example, 5 Â 109/l), but rather on the risk of developing 2005 MBL diagnostic criteria strongly relates to TFS.19,20 As clinically relevant outcomes such as disease-related symptoms, discussed earlier, the risk of progression requiring CLL-specific infection, need for treatment or death. treatment among individuals with MBL is 1–2% per year18,19,25 compared with 5–7% per year for individuals with Rai stage 0 CLL.19,27 Although this difference appears subtle, it equates to a 10-year risk of requiring treatment of 7–14% for individuals with Is CLL always preceded by MBL? MBL compared with 50–70% for patients with Rai stage 0 CLL. Another important question is whether all CLL cases are Consistent with these clinical findings, the 2008 update to the preceded by MBL, or if some (for example, biologically CLL diagnostic criteria incorporated a B-cell threshold of aggressive) CLL cases develop de novo. This is of importance 5 Â 109/l used in the 2005 MBL criteria.26 because if MBL consistently precedes CLL researchers could Although these changes have anchored the diagnosis of CLL develop prospective studies to uncover the biologic mechanisms to clinical outcome, further investigation is needed to determine of CLL leukemiogenesis. To address this question, Landgren how best to distinguish between CLL and MBL. First, B-cell et al.28 recently conducted a prospective cohort study based on count appears to relate to TFS and OS as a continuous 77 469 healthy adults who were enrolled in the nationwide, variable19,20 and recent studies suggest higher B-cell thresholds population-based, US Prostate, Lung, Colorectal and Ovarian (for example, 11 Â 109/l) may not only stratify TFS but also Cancer Screening Trial.28 The investigators identified 45 predict OS.20 Second, although these studies have shown the participants who were subsequently diagnosed with CLL during ability of the B-cell count to stratify survival in patients with a the period of longitudinal observation who had a pre-diagnostic clonal population of CLL phenotype, they have not bench- peripheral blood sample available for analysis. marked the outcome of these individuals to the general Using six-color flow cytometry and IGHV gene analysis by population. Conceptually, the expected survival of individuals reverse transcriptase PCR, the investigators found evidence of with a precursor state to malignancy (for example, adenomatous MBL predating the CLL diagnosis in 44 patients (98%).28 It is colon polyps) should be the same as that of the general noteworthy that MBL was present up to 6.4 years before CLL population unless they go on to develop the at-risk disease diagnosis in these individuals. In 41 patients (91%), the clone (for example, colon cancer). Although B-cell count is clearly was confirmed by both analysis methods.28 The IGHV gene established as a prognostic factor in patients with MBL, whether mutation status was determined in 35 of 45 MBL clones (78%). or not it can be used to identify those individuals with a Of these, 16 (46%) were IGHV3 subgroup genes (including circulating B-cell clone whose OS is shorter than unaffected 6 (17%) IGHV3–23 genes) and 9 (26%) were IGHV4 subgroup individuals in the general population has not yet been genes (including 4 (11%) IGHV4–34 genes).28 The distribution determined. Third, although the diagnosis of CLL and MBL of mutated clones as compared with unmutated clones was should be based in part on clinical outcomes, yet to be similar regardless of the time at which the blood samples was discovered biologic characteristics could be an equally im- obtained in relationship to subsequent CLL diagnosis. Although portant consideration. The 5% prevalence of MBL in the general the number of IGHV unmutated samples was small, three of population over age 60 coupled with the markedly lower eight IGHV unmutated clones were present more than 3 years prevalence of CLL is consistent with the multi-hit hypothesis of before the CLL diagnosis, with two detectable 5 years before. human malignancy and suggests additional biologic events must Thus, this study suggests that virtually all cases of CLL, including occur for MBL to progress to CLL. Identification of the biologic both mutated and unmutated IGHV cases, are preceded by events that related to progression could lead to incorporation of MBL, which is often present for years before clinical CLL these characteristics in the classification system as well as the diagnosis.28 development of early intervention strategies.

Leukemia Monoclonal B-cell lymphocytosis TD Shanafelt et al 517 Table 3 Distinguishing between MBL, CLL and SLL

Clonal B cells of Peripheral blood B-cell Lymphadenopathy or Bone marrow findings CLL phenotype count o5 Â 109/l hepatosplenomegaly

MBL Yes Yes No Marrow biopsy is not required as part of either the MBL or CLL diagnostic evaluationa SLL Yes Yes Yes CLL Yes No Yes or no Abbreviations: CLL, chronic lymphocytic leukemia; MBL, monoclonal B-cell lymphocytosis; SLL, small lymphocytic lymphoma. aThe limited available data suggest that nearly all patients with clinically identified MBL will have some degree of bone marrow involvement if marrow biopsy is performed with a median of 10–20% involvement.27,32 More extensive marrow involvement (70–80%) has been observed in some individuals with MBL but is not clearly related to risk of progression.32

Clinical management of MBL recommend annual follow-up by a hematologist–oncologist with a . Recommendations for Evaluation and Follow-up At this time we lack tools to predict, which MBL cases will There exists limited data on the clinical management of patients progress to CLL. In small series, surface expression of CD38 has with MBL. The great majority of individuals with MBL been correlated with the future need for therapy in two19,27 of (approximately 85%) are currently identified as part of the three studies.18 Some (IGHV genes mutations, CD49d, FISH) but clinical evaluation of an abnormal blood count, most commonly not all (ZAP-70, beta-2 microglobulin) prognostic parameters lymphocytosis. These individuals with clinical MBL should used to predict TFS in CLL were also reported to be useful undergo a complete evaluation by a hematologist–oncologist prognostic parameter for clinical MBL patients in one small that includes a detailed family history (given the association series.27 Currently, there is insufficient evidence to routinely between CLL and MBL), a review for B-type symptoms, and a recommend such prognostic testing for individuals with MBL complete physical examination with comprehensive lymph outside of investigational protocols. node examination. In contrast to the measurable risk of progression in patients The most critical step in the clinical management of with clinical CLL-like MBL, progression among patients with individuals with MBL is to insure the correct diagnosis. population-screening CLL-like MBL is exceedingly rare in the Individuals with MBL should be distinguished from patients investigators’ experience. As discussed in the previous sections, with subtle manifestations of NHL. In the absence of constitu- there is emerging data that the biology of population-screening tional symptoms (, night sweats, fatigue and weight loss), MBL differs from that of CLL8,14 and clinical MBL and that individuals with CLL-like MBL can be distinguished from those population-screening MBL can be identified in up to 20% of the with SLL based on the absence of palpable lymphadenopathy or general population using highly sensitive assays.9 Therefore, we organomegaly on physical examination (Table 3). Although believe that individuals with population-screening MBL likely marrow biopsy is not required, nearly all patients with clinically do not have a risk of CLL that is substantially higher than that of identified MBL will have some degree of bone marrow the general population. Such patients do not require formal involvement if biopsy is performed.27,32 Although the median evaluation by a hematologist or surveillance beyond an annual percent marrow involvement in clinical MBL appears to be examination by a primary care provider with complete blood between 10 and 20%, more extensive involvement is observed count. in some individuals.27,32 In the series from Mayo Clinic, The risk of progression among patients with atypical-CLL- although 3 out of 46 (6.5%) individuals with MBL had extensive phenotype MBL or non-CLL-phenotype MBL is less well defined. marrow involvement on biopsy (70 to 80%) none of these 3 For rare individuals with a phenotype and cytogenetic studies individuals had required treatment on up to 5-year follow-up.32 suggestive of mantle cell lymphoma or another aggressive NHL For individuals with an atypical-CLL-phenotype MBL or non- subtype but without adenopathy on computed tomography CLL-phenotype MBL, a more thorough evaluation is required scanning or substantial marrow involvement on marrow biopsy, because standard staging for NHL requires computed tomo- we suggest clinical follow-up every 3–6 months with computed graphy scanning (chest, abdomen and pelvis) and bone marrow tomography imaging at least every 6 months.34 For those biopsy.33 Individuals with radiographic evidence of adenopathy patients with atypical-CLL-phenotype MBL or non-CLL-pheno- are best classified as having NHL with the sub-classification type MBL whose immunophenotype is consistent with a more (for example, marginal zone lymphoma, , indolent NHL subtype (for example, marginal zone lymphoma, mantle cell lymphoma) based on immunophenotyping and, follicular lymphoma), follow-up by a hematologist–oncologist wherein relevant, cytogenetic analysis (for example, FISH every 6–12 months is recommended. The frequency of follow- evaluating for t(11;14)) or immunohistochemistry (for example, up imaging requires clinical judgment and must be balanced cyclin D1 staining).33 In appropriate circumstances, serum/urine with the risks associated with radiation exposure.35 protein electrophoresis to evaluate for lymphoplasmacytic Some have questioned whether or not it is ethical to inform lymphoma should be pursued (Table 4). individuals with population-screening MBL identified as part of As detailed, the average risk of progression requiring therapy research studies of their test results given the absence of among individuals with clinical CLL-like MBL appears to be evidence that population-screening MBL predicts a clinically approximately 1–2% per year.18,19,25 On the basis of this data, important outcome (for example, it lacks clinical validity as a we reassure individuals with CLL-like MBL identified in clinical predictor for developing CLL or other NHL subtypes) or that practice that they are at low risk for developing CLL, counsel knowledge regarding population-screening MBL improves the them regarding symptoms they should watch for (lymph- subjects well-being (for example, it lacks clinical utility).36 It adenopathy, fevers, night sweats, fatigue, weight loss), and can, in fact, be argued that knowledge of population-screening

Leukemia Monoclonal B-cell lymphocytosis TD Shanafelt et al 518 Table 4 Recommendations for evaluation and follow-up of MBL in routine practice

Recommendations Population-screeninga Clinical a Atypical or non-CLL-like MBL CLL-like MBL CLL-like MBL identified in a clinical setting

Diagnostic evaluation Historyb Yes Yes Yes Physical examc Yes Yes Yes Immunophenotype of lymphocytes Yes Yes Yes CBC with differential Yes Yes Yes FISH testing with probe for t(11;14) No No Yesd CT scan chest/abd/pelvis No No Yes Bone marrow biopsy No No Yes CLL prognostic testing No No No

Counseling and follow-up Patient counseling on symptoms to watch forb Yes Yes Yes Risk of progression requiring therapy Lowe 1–2%/year Undefined Historyb Routine medical care annual 3–12 monthsf Physical examc Routine medical care annual 3–12 monthsf CBC with differential Annual 6–12 months 6–12 monthsf CT scan chest/abd/pelvis No No Clinical judgementf Abbreviations: CBC, complete blood count; CLL, chronic lymphocytic leukemia; CT, computed tomography; FISH, fluorescence in situ hybridization; MBL, monoclonal B-cell lymphocytosis; NHL, non-Hodgkin lymphoma. aCLL-phenotype MBL identified as part of clinical evaluation of lymphocytosis is referred to as clinical CLL-like MBL while CLL-phenotype MBL identified in research studies of patients with normal lymphocyte counts is referred to as population-screening CLL-like MBL. bFocus on constitutional symptoms (fevers, night sweats, weight loss and fatigue). cFocus on lymph node evaluation and assessment for hepatosplenomegaly. dFor patients with atypical phenotype CLL who are CD5+ but CD23– FISH testing with a probe to detect t(11;14) characteristic of mantle cell lymphoma should be performed. eAlthough limited data are available, progression among patients with population-screening CLL-like MBL is rare in the authors’ experience. Because several series suggest that B-cell count is related to clinical outcome in individuals with MBL,18–20 the risk of progression for individuals with population-screening CLL-like MBL is thought to be less than that of individuals with Clinical CLL-like MBL. fFor the rare individuals fulfilling the criteria for MBL who have a phenotype and cytogenetic studies suggestive of mantle cell lymphoma or another aggressive NHL subtype, we suggest clinical follow-up every 3–6 months with CT imaging at least every 6 months. For those patients with atypical- CLL-phenotype MBL or non-CLL-phenotype MBL whose immunophenotype is consistent with a more indolent NHL subtype, follow-up every 6–12 months is recommended and the frequency of follow-up imaging requires clinical judgment.

MBL may worsen an individuals well-being by causing undue discussions with their family. Similarly, MBL should not be anxiety and/or exposing them to possible discrimination by referred to as ‘pre-leukemia’ or ‘pre-CLL’ as the majority of employers or life/health insurers.36 Although there is consensus patients with clinical MBL will never develop symptoms or that research subjects should be informed of new information require CLL-directed therapy. gained from their participation,37 whether the presence of The distinction between MBL and CLL also has several other population-screening MBL constitutes ‘meaningful information’ practical ramifications. First, MBL does not currently have a can only be defined through ongoing and future studies.36 specific ICD-9 code for billing purposes, which forces clinicians to choose between coding individuals with MBL as lympho- cytosis (ICD-9 code 288.8) or CLL (ICD-9 code 204.1) either of Other clinical issues which creates potential problems. Although the classification of As flow cytometry is more widely used, more individuals with a ‘lymphocytosis’ correctly identifies the non-malignant nature mild absolute lymphocytosis will have a flow cytometric of MBL, it may create difficulties with insurance coverage for evaluation. In many laboratories, identification of a CLL-like evaluation and care provided by a hematologist or diagnostic population leads to a report stating ‘a clonal population testing, particularly in some European countries. In contrast, in consistent with a diagnosis of CLL or small lymphocytic the US health-care system, a diagnosis of CLL could have lymphoma has been identified’. This language may need to be implications for insurability as a pre-existing condition when revised to include the possibility that the findings represent MBL. individuals change their health-care provider and may affect an Owing to recent updates to the diagnostic criteria for CLL,26,30 individual’s ability to obtain life insurance. This distinction also many individuals fulfilling the criteria for a diagnosis of clinical has repercussions to insurance policies that provide specific MBL will have previously been given a diagnosis of CLL.20,31 On benefits after a diagnosis of ‘cancer’ causing some patients to account of the significant differences in risk and clinical lobby for the CLL code rather than the lymphocytosis code. outcome,20,27,31 it is important to inform such patient that the These challenges illustrate some of the reasons why a new correct diagnosis is MBL, provide education that MBL is not a ICD-9 code specific to MBL is needed. malignant diagnosis, and discuss the appropriate monitoring and Another challenge encountered in clinical practice is the use follow-up. With time and counseling, most patients will of cellular blood products donated by individuals with MBL. understand that clinical MBL and CLL fall on a continuum. Although one study found the frequency of MBL in blood donors The distinction that an MBL diagnosis indicates a higher lifetime to be quite low,41 the study used a relatively insensitive risk of developing CLL, rather than being a diagnosis screening assay that likely underestimated the true prevalence of leukemia, avoids the unnecessary distress associated with of MBL among blood donors. Further, there are no data a leukemia diagnosis38–40 and may aid the individual in regarding clinical outcomes from MBL donor-derived blood

Leukemia Monoclonal B-cell lymphocytosis TD Shanafelt et al 519 Table 5 Key areas for future MBL research it is identified during the diagnostic evaluation of lymphocytosis or incidentally discovered through screening studies using (I) Identification of epidemiologic factors associated with development highly sensitive detection methods. Additional research into of MBL the epidemiology, biology and natural history of MBL is needed K Genetics (Table 5). The majority of individuals with MBL will not develop K Health behaviors a hematologic malignancy. K Exposures

(II) Clinical outcome of low-count MBL K CLL-like MBL Conflict of interest K Atypical- and non-CLL-phenotype MBL K Prognostic markers The authors declare no conflict of interest.

(III) Identification of clinical and biologic parameters that allow optimal classification of patients with small B-cell clones (MBL vs CLL vs SLL) Acknowledgements K Characteristics of the clone K Characteristics of the host/environment This work was supported by grants from the National Cancer Institute (NCI CA 113408; T Shanafelt), Italian Association for (IV) Identification of the biologic events related to progression of MBL to F CLL or other NHL Cancer Research AIRC, Milano, Italy (P Ghia), Leukaemia K Biologic events in the clonal B cells Research Fund, UK (A Rawstron). K Biologic events/interactions related to the host/environment K Genetic characteristics of the clone K Germline genetic characteristics of the host References K Behaviors K Exposures 1 Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ. Cancer statistics, 2009. CA Cancer J Clin 2009; 59: 225–249. (V) Studies exploring a potential tissue equivalent of MBL 2 Vogt RF, Shim YK, Middleton DC, Buffler PA, Campolucci SS, Abbreviations: CLL, chronic lymphocytic leukemia; MBL, monoclonal Lybarger JA et al. Monoclonal B-cell lymphocytosis as a biomarker B-cell lymphocytosis; NHL, non-Hodgkin lymphoma; SLL, small in environmental health studies. Br J Haematol 2007; 139: 690–700. lymphocytic lymphoma. 3 Shim YK, Vogt RF, Middleton D, Abbasi F, Slade B, Lee KY et al. Prevalence and natural history of monoclonal and polyclonal B-cell lymphocytosis in a residential adult population. Cytometry B products to inform the decision of whether individuals with Clin Cytom 2007; 72: 344–353. clinical MBL can safely serve as donors. Currently, individuals 4 Rawstron A, Green M, Kuzmicki A, Kennedy B, Fenton J, Evans P with an underlying malignancy, including CLL, cannot donate et al. Monoclonal B lymphocytes with the characteristics of ‘indolent’ chronic lymphocytic leukemia are present in 3.5% of blood, bone marrow, umbilical cord blood or solid organs for adults with normal blood counts. Blood 2002; 100: 635–639. transplantation. As population-screening MBL can be identified 5 Ghia P, Prato G, Scielzo C, Stella S, Geuna M, Guida G et al. in up to 20% of the adult population when evaluated using Monoclonal CD5+ and CD5- B-lymphocyte expansions are frequent sensitive methods,9 this issue could have profound affects on the in the peripheral blood of the elderly. Blood 2004; 103: 2337–2342. supply of organ and blood products and warrants clinical 6 Marti GE, Rawstron AC, Ghia P, Hillmen P, Houlston RS, Kay N studies. At present, it is premature to recommend screening et al. Diagnostic criteria for monoclonal B-cell lymphocytosis. Br J Haematol 2005; 130: 325–332. blood or solid organ donors for MBL if the donor’s complete 7 Rawstron AC, Kennedy B, Evans PA, Davies FE, Richards SJ, blood count is normal. Haynes AP et al. Quantitation of minimal disease levels in chronic A final challenge with regard to MBL patients donating lymphocytic leukemia using a sensitive flow cytometric assay cellular products pertains to stem cell transplant. Although the improves the prediction of outcome and can be used to optimize role of allogenic stem cell transplant as a treatment for patients therapy. Blood 2001; 98: 29–35. with CLL continues to be refined, it is a proven and routinely 8 Dagklis A, Fazi C, Sala C, Cantarelli V, Scielzo C, Massacane R 42–44 et al. The immunoglobulin gene repertoire of population CLL-like used therapy for selected CLL patients. As approximately MBL is different from CLL: diagnostic implications for clinical 10,11 10% of CLL cases occur in a familial pattern and the monitoring. Blood 2009; 114: 26–32. prevalence of MBL is increased in such CLL families,12,13 the 9 Nieto WG, Almeida J, Romero A, Teodosio C, Lopez A, Henriques question of whether relatives preparing to serve as a stem cell AF et al. Increased frequency (12%) of circulating chronic donor for CLL patients undergoing allogeneic transplant should lymphocytic leukemia-like B-cell clones in healthy subjects using undergo screening for MBL is a practical issue.36 There is not a highly sensitive multicolor flow cytometry approach. Blood 2009; 114: 33–37. currently consensus on whether or not matched relatives with 10 Goldin L, Pfeiffer R, X XL, Hemminki K. Familial risk of MBL should be disqualified as donors. For many patients no lymphoproliferative tuors in families of patients with chronic alternative donor may be available and, even if available, the lymphocytic leukemia: results from the swedish family-cancer use of a matched unrelated donor could expose the patients to database. 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