PhD Andy C Rawstron
email: andy.rawstron@nhs.net

Occult B-lymphoproliferative Disorders

Keywords: Chronic Lymphocytic Leukaemia, Non-Hodgkin Lymphoma, Monoclonal B-cell Lymphocytosis Chronic Lymphocytic Leukemia (CLL), Non-Hodgkin Lymphoma, Monoclonal B-cell Lymphocytosis (MBL)

published or not. The documents may come

Introduction

Monoclonal B-cell Lymphocytosis (MBL) is a classification that has arisen largely because of the increasing sensitivity of diagnostic techniques, particularly for Chronic Lymphocytic Leukaemia (CLL). Over the past century the incidence of CLL has more than doubled [START_REF] Call | Incidence of chronic lymphocytic leukemia in Olmsted County, Minnesota, 1935 through 1989, with emphasis on changes in initial stage at diagnosis[END_REF][START_REF] Molica | What is changing in the natural history of chronic lymphocytic leukemia?[END_REF] largely through routine usage of haematology analysers. This change in incidence has been associated with prolonged survival of individuals diagnosed with the disease, largely through increased detection at the asymptomatic early stage disease which is associated with a lesser reduction in overall survival compared to more advanced stage disease [START_REF] Abrisqueta | Improving survival in patients with chronic lymphocytic leukemia (1980-2008): the Hospital Clinic of Barcelona experience[END_REF][START_REF] Molica | What is changing in the natural history of chronic lymphocytic leukemia?[END_REF]. The ability to detect CLL at an increasingly early stage has not led to improvements in overall survival for such individuals (3) and they experience significant anxiety as a result of the diagnosis, surprisingly greater even than for other cancer diagnoses, because of the uncertain outcome [START_REF] Shanafelt | Quality of life in chronic lymphocytic leukemia: an international survey of 1482 patients[END_REF]. The situation is less clear for other B-lymphoproliferative disorders where diagnosis may be more dependent on the presence of palpable lymphadenopathy, but the incidence rates for non-Hodgkin lymphoma are also reported to have doubled since the mid-1970s [START_REF]Browse the SEER Cancer Statistics Review[END_REF].

In addition to increased surveillance and investigation, the detection of Blymphoproliferative disorders is also affected by the diagnostic technology available.

Up until the start of this millennium, most diagnostic laboratories were only able to access flow cytometers with one or two colours which offered a sensitivity of detection that was similar to immunohistochemistry. Even using such basic cytometry it is possible to detect monoclonal B-cells in nearly 1% of adults [START_REF] Vogt | Monoclonal B-cell lymphocytosis as a biomarker in environmental health studies[END_REF]. However, the development of cytometers with four or more colours provided a major increase in the potential sensitivity of detection with the ability to routinely detect, characterise and quantify phenotypically abnormal B-cells representing as few as 1 cell in 10,000 leucocytes [START_REF] Rawstron | Quantitation of minimal disease levels in chronic lymphocytic leukemia using a sensitive flow cytometric assay improves the prediction of outcome and can be used to optimize therapy[END_REF][START_REF] Rawstron | International standardized approach for flow cytometric residual disease monitoring in chronic lymphocytic leukaemia[END_REF]. When applied to the general population, such techniques could detect abnormal B-cell populations in 3-5% of adults with normal blood counts and no history of cancer. [START_REF] Rawstron | Monoclonal B lymphocytes with the characteristics of "indolent" chronic lymphocytic leukemia are present in 3.5% of adults with normal blood counts[END_REF][START_REF] Ghia | Monoclonal CD5+ and CD5-B-lymphocyte expansions are frequent in the peripheral blood of the elderly[END_REF]. The availability of such technology in routing laboratories, coupled with a greater propensity for referring individuals with a slight lymphocytosis for laboratory investigations, led to an increasing number of individuals being identified as having circulating monoclonal B-cells but not meeting the criteria for a lymphoproliferative disorder. Such individuals do not have an overt leukaemia, the likelihood of developing progressive disease seems low but there was a lack of information concerning outcome in part because there was a highly variable approach to diagnosis and monitoring in this setting [START_REF] Marti | Diagnostic criteria for monoclonal B-cell lymphocytosis[END_REF].

Proposal of diagnostic criteria for Monoclonal B-cell Lymphocytosis

The term Monoclonal B-cell Lymphocytosis (MBL) was introduced to provide a framework for understanding the clinical requirements of such individuals. MBL describes the presence of a population of monoclonal B-cells in the peripheral blood of an individual with no other features of a B-lymphoproliferative disorder . The absolute B-cell count in MBL is less than 5,000/µL (5 x 10 9 /L) and the majority of reported cases have a phenotype consistent with CLL. Individuals with MBL should have no symptoms of a lymphoproliferative disorder such as drenching night sweats or unexplained fever, fatigue or weight loss and no palpable lymphadenopathy or splenomegaly suggestive of a lymphoproliferative disorder [START_REF] Marti | Diagnostic criteria for monoclonal B-cell lymphocytosis[END_REF]. The diagnostic criteria for MBL are shown in Table 1.

These diagnostic criteria were intended to allow assessment of any clinical significance and investigate the underlying pathology of B-lymphoproliferative disorders by identifying biological factors present at the earliest stages of disease.

1) Detection of a monoclonal B-cell population in the peripheral blood with i) overall kappa:lambda ratio >3:1 or <0.3:1, or ii) greater than 25% of B cells lacking or expressing low level surface immunoglobulin or iii) a disease-specific immunophenotype.

2) Repeat assessment should demonstrate that the monoclonal B-cell population is stable over a 3-month period.

3) Exclusion criteria

MBL in population studies: association between prevalence and assay sensitivity

Early studies detect MBL in less than 1% of the population Between 1991 and 1994 the National Center for Environmental Health in the US investigated the potential health impact of living around hazardous waste sites [START_REF] Vogt | Monoclonal B-cell lymphocytosis as a biomarker in environmental health studies[END_REF].

The study protocol included an immune biomarker panel with basic lymphocyte immunophenotyping but not B-cell Kappa:Lambda analysis. An abnormal B-cell phenotype was observed in 11 of 1926 participants (age range 47 to 72), of which 10/11 had an elevated total B-cell. Further tests confirmed an abnormal phenotype with light-chain restriction. Two additional cases with a B-cell clone were discovered during follow-up studies [START_REF] Shim | Prevalence and natural history of monoclonal and polyclonal B-cell lymphocytosis in a residential adult population[END_REF]. Using this approach the overall prevalence was reported as 9/1499 (0.6%) of individuals. One case was diagnosed with CLL shortly after first follow-up and died with progressive disease two years after the abnormal Bcell population was detected. A second case was subsequently diagnosed with Waldenström's macroglobulinemia and had stable disease at last follow-up. Studies using basic flow cytometry in anonymised samples from blood donors in the US

Disease-specific assays increase the reported prevalence of MBL

The increasing efficacy of treatment in CLL led to the development of assays that could detect residual disease in a background of polyclonal B-cells with a higher specificity and sensitivity than basic flow cytometry [START_REF] Vuillier | Evaluation of residual disease in B-cell chronic lymphocytic leukemia patients in clinical and bone-marrow remission using CD5-CD19 markers and PCR study of gene rearrangements[END_REF][START_REF] Rawstron | Quantitation of minimal disease levels in chronic lymphocytic leukemia using a sensitive flow cytometric assay improves the prediction of outcome and can be used to optimize therapy[END_REF][START_REF] Vela | CD79b expression in B cell chronic lymphocytic leukemia: its implication for minimal residual disease detection[END_REF][START_REF] Esteve | Stem cell transplantation for chronic lymphocytic leukemia: different outcome after autologous and allogeneic transplantation and correlation with minimal residual disease status[END_REF]. The application of such assays to population studies resulted in a large increase in the reported prevalence of CLL-type MBL.

In the UK [START_REF] Rawstron | Monoclonal B lymphocytes with the characteristics of "indolent" chronic lymphocytic leukemia are present in 3.5% of adults with normal blood counts[END_REF], samples from 425 male and 485 female hospital outpatients, median age 65 (range 40-90) with a normal blood count and no history or suspicion of cancer were tested by four-color flow cytometry. Cells with a CLL phenotype were detected in 3.5% of total with a male:female ratio of 2.2:1. The prevalence increased with age, from 2.1% in the individuals between 40 and 60 years old to 5.0% for individuals over 60 (chi-square P=0.01). The absolute numbers of CLL phenotype cells were low (median 0.013, range 0.003 to 1.46 x 10 9 /L) and represented a minor proportion of total B-lymphocytes in most cases (median of 11%, range 3 -95%). In addition a CD5-MBL was detected due to a perturbation of kappa:lambda ratio in a further nine (1.0%) of the 910 of individuals. These were mostly elderly individuals (median age 78 years old, range 49 -88) [START_REF] Rawstron | Monoclonal B lymphocytes with the characteristics of "indolent" chronic lymphocytic leukemia are present in 3.5% of adults with normal blood counts[END_REF]. Similar results were reported in a study involving a rural community outside Turin [START_REF] Ghia | Monoclonal CD5+ and CD5-B-lymphocyte expansions are frequent in the peripheral blood of the elderly[END_REF] with samples from 269 females and 231 males (mean age 74, range 65-98 years) referred for common routine blood test (e.g. blood glucose, blood lipids). All individuals included in the study had a normal blood cell count, with no evidence of lymphocytosis and no history or suspicion of malignancy at the time of the routine blood test. Using a sensitive disease-specific protocol, CLL phenotype cells were detected in 5.5%, of individuals above 65 years of age, closely resembling the 5% frequency reported among the elderly (> 60 years) in the UK study [START_REF] Rawstron | Monoclonal B lymphocytes with the characteristics of "indolent" chronic lymphocytic leukemia are present in 3.5% of adults with normal blood counts[END_REF]. CLL phenotype cells represented a small proportion of total CD19+ B-cells (mean 1.8%, range 0.7% -4%) The prevalence of monoclonal B-cells increased with age, being higher in individuals above 75 years of age and a male prevalence was manifest, especially among the latter age group. A CD5-monoclonal B-cell population was detected in 1.4%. By increasing the number of parameters and events measured, the prevalence of CLLtype MBL showed almost a ten-fold increase. More recently the reported prevalence for CLL-type MBL increased even further in a report from the Primary Health Care Group of Salamanca for the Study of MBL. This study utilized the highest sensitivity flow cytometry approaches available with 10-parameter analysis of 5 million events. CLL-phenotype cells were detectable in more than 1 in 5 individuals over 60 years old 27. The percentage of aberrant/clonal B cells was below the maximum sensitivity of the latter approaches (<0.01%) in more than half of the cases (62%) although it should be noted that clonality was only confirmed by additional methods in 18 cases, all except one with >0.01% aberrant/clonal B-cells. [START_REF] Nieto | Increased frequency (12%) of circulating chronic lymphocytic leukemialike B-cell clones in healthy subjects using a highly sensitive multicolor flow cytometry approach[END_REF].

The biological relationship between MBL and associated Blymphoproliferative disorders

The presence of a small population of monoclonal B-cells in such a high proportion of the general population has understandably led to the question of whether such cells are genuinely neoplastic or simply represent a small reactive expansion. For CLL-type MBL there is evidence on several levels that the cells are neoplastic. Extensive studies of protein expression confirm that the phenotype of the abnormal B-cells is consistent with CLL and different to any other normal B-cell population even for cases with very low levels of abnormal B-cells [START_REF] Rawstron | The biological and clinical relationship between CD5+23+ monoclonal B-cell lymphocytosis and chronic lymphocytic leukaemia[END_REF][START_REF] Rawstron | Chronic lymphocytic leukaemia (CLL) and CLL-type monoclonal B-cell lymphocytosis (MBL) show differential expression of molecules involved in lymphoid tissue homing[END_REF]. Chromosomal abnormalities consistent with CLL, notably the 13q14 deletion affecting mir-15a/16-1 which is reported to cause CLL [START_REF] Klein | The DLEU2/miR-15a/16-1 Cluster Controls B Cell Proliferation and Its Deletion Leads to Chronic Lymphocytic Leukemia[END_REF], are detected in CLL-type MBL even in cases where the neoplastic B-cells represent <10 cells per µL [START_REF] Rawstron | Monoclonal B-cell lymphocytosis and chronic lymphocytic leukemia[END_REF][START_REF] Shanafelt | Natural History of Individuals With Clinically Recognized Monoclonal B-Cell Lymphocytosis Compared With Patients With Rai 0 Chronic Lymphocytic Leukemia[END_REF][START_REF] Nieto | Increased frequency (12%) of circulating chronic lymphocytic leukemialike B-cell clones in healthy subjects using a highly sensitive multicolor flow cytometry approach[END_REF][START_REF] Lanasa | Family-Associated Monoclonal B Lymphocytosis Is Commonly Oligoclonal and Expresses Markers Associated with Adverse Risk in CLL[END_REF]. The underlying genetic factors responsible for development of CLL also result in an increased susceptibility to CLL-type MBL as shown in CLL families [START_REF] Rawstron | Inherited predisposition to CLL is detectable as subclinical monoclonal B-lymphocyte expansion[END_REF][START_REF] Marti | B-cell monoclonal lymphocytosis and B-cell abnormalities in the setting of familial B-cell chronic lymphocytic leukemia[END_REF] [START_REF] Amato | Cytogenetic aberrations and immunoglobulin VH gene mutations in clinically benign CD5-monoclonal B-cell lymphocytosis[END_REF][START_REF] Nieto | Non-CLL-like monoclonal B-cell lymphocytosis in the general population: Prevalence and phenotypic/genetic characteristics[END_REF].

The clinical relationship between MBL and associated Blymphoproliferative disorders

There are several studies which demonstrate a clinical association between CLL-type MBL and clinical disease. Pre-diagnostic monoclonal B-cell populations are detectable up to 6.4 years prior to diagnosis of CLL in most (44/45) cases [START_REF] Landgren | B-cell clones as early markers for chronic lymphocytic leukemia[END_REF].

However, only a limited number of cases with CLL-type MBL show progressive disease, and this is largely limited to cases which present with an absolute lymphocytosis. Three studies have evaluated the impact of the neoplastic B-cell count on risk of developing progressive lymphocytosis, treatment-free survival and overall survival [START_REF] Rawstron | Monoclonal B-cell lymphocytosis and chronic lymphocytic leukemia[END_REF][START_REF] Shanafelt | Bcell count and survival: differentiating chronic lymphocytic leukemia from monoclonal B-cell lymphocytosis based on clinical outcome[END_REF][START_REF] Rossi | The prognosis of clinical monoclonal B cell lymphocytosis differs from prognosis of Rai 0 chronic lymphocytic leukaemia and is recapitulated by biological risk factors[END_REF]. In the Leeds study 185 CLL-type MBL cases were monitored for median of 80 months and approximately 1.1% per year required treatment for disease progression [START_REF] Rawstron | Monoclonal B-cell lymphocytosis and chronic lymphocytic leukemia[END_REF]. In the Mayo Clinic studies, 302 CLL-type MBL cases were monitored for a median 18 months with approximately 1.4% per year requiring treatment for disease progression [START_REF] Shanafelt | Brief report: natural history of individuals with clinically recognized monoclonal B-cell lymphocytosis compared with patients with Rai 0 chronic lymphocytic leukemia[END_REF]. In the Italian study, 123 CLL-type MBL cases were monitored for a median of 43 months with 4% per year requiring treatment in the first seven years with very little disease progression occuring thereafter [START_REF] Rossi | The prognosis of clinical monoclonal B cell lymphocytosis differs from prognosis of Rai 0 chronic lymphocytic leukaemia and is recapitulated by biological risk factors[END_REF].

In the studies from the UK and Italy a B-cell count at presentation below 1,200 per mm 3 or 1,900 per mm 3 respectively predicted a subsequently stable lymphocyte count, whereas a presentation B-cell count above 3,700 per mm 3 or 4,000 per mm 3 respectively predicted a higher probability of a subsequently rising lymphocyte count F o r P e e r R e v i e w [START_REF] Rawstron | Monoclonal B-cell lymphocytosis and chronic lymphocytic leukemia[END_REF][START_REF] Rossi | The prognosis of clinical monoclonal B cell lymphocytosis differs from prognosis of Rai 0 chronic lymphocytic leukaemia and is recapitulated by biological risk factors[END_REF]. The B-cell count predicted treatment-free survival in the Mayo series [START_REF] Shanafelt | Brief report: natural history of individuals with clinically recognized monoclonal B-cell lymphocytosis compared with patients with Rai 0 chronic lymphocytic leukemia[END_REF] but not in the UK and Italian studies. These differences may reflect variations due to the small numbers of cases with disease progression, but the UK and Italian studies had longer follow-up and it may be that a higher B-cell count predicts an increased risk of disease progression requiring treatment only in the first few years of follow-up.

There is some evidence that an increased presentation B-cell count as a continuous variable is associated with poorer overall survival whereas an increased T-cell count is associated with an improved overall survival [START_REF] Rawstron | Monoclonal B-cell lymphocytosis and chronic lymphocytic leukemia[END_REF][START_REF] Shanafelt | Bcell count and survival: differentiating chronic lymphocytic leukemia from monoclonal B-cell lymphocytosis based on clinical outcome[END_REF]. In addition to the risk of progression requiring therapy, it is possible that individuals with CLL-type MBL who have higher CLL cell counts may have greater suppression of normal B-cell activity resulting in an increased susceptibility to infection. In the Leeds series of patients with clinically identified MBL (i.e. all with B-cell counts below 5,000 per mm 3 ), a B-cell count above 3.2 x 10 9 /L was the optimal cut-off point for predicting overall survival [START_REF] Rawstron | Monoclonal B-cell lymphocytosis and chronic lymphocytic leukemia[END_REF]. In the Mayo clinic series, which included both patients with MBL as well as Rai stage 0 CLL, a B-cell threshold of 11 x 10 9 /L optimally predicted overall survival [START_REF] Shanafelt | Bcell count and survival: differentiating chronic lymphocytic leukemia from monoclonal B-cell lymphocytosis based on clinical outcome[END_REF].

Outcome for population studies is generally not known, either because of limited follow-up time or because the studies were performed in an anonymised fashion for ethical reasons. Initial data indicates that the five-year outcome, in terms of overall survival and risk of infections, is not different for individuals with a detectable MBL population but a normal blood count. A proportion of cases with CLL-type MBL, approximately 1% per year, show a rising CLL cell count and are referred for investigation of a lymphocytosis but otherwise the presence of a small population of monoclonal B-cells in a background of polyclonal B-cells does not appear to have any clinical impact [START_REF] Bennett | Five-Year Follow-up of Monoclonal B-Cell Lymphocytosis (MBL) in Individuals with a Normal Blood Count: Expansion of the Abnormal B-Cell Compartment but No Progressive Disease[END_REF].

Clinical series of CD5-negative MBL cases are infrequent. In a report from Toronto, 7 cases of CD5-negative MBL with an absolute lymphocytosis were shown to have a stable condition with little change in the B-cell count after several years of follow-up

The cases were clearly not reactive B-cell expansions as five of six evaluable cases had clonal cytogenetic abnormalities including two cases with isochromosome 17q resulting in loss of p53 [START_REF] Amato | Cytogenetic aberrations and immunoglobulin VH gene mutations in clinically benign CD5-monoclonal B-cell lymphocytosis[END_REF]. The lack of large clinic series for CD5-negative MBL may reflect the fact that most non-Hodgkin lymphoma have little peripheral blood involvement and therefore detection of circulating cells usually only occurs if there is significant tissue disease. In addition to the series of 362 cases of CLL-type MBL

Monoclonal B-cell populations in the bone marrow

The capability to detect small monoclonal B-cell populations in a large proportion of individuals with normal blood counts and no history of malignancy has the potential to cause significant problems in diagnostic laboratories as such technology becomes more widely available. The current routine approach at the HMDS laboratory for screening peripheral blood, bone marrow and lymph node samples for the presence of an abnormal B-cell population involves acquisition of 100,000 events using 8parameter flow cytometry. This is equivalent to the mid-sensitivity population studies performed at the turn of the century, which would potentially allow detection of MBL in approximately 3% of otherwise healthy adults over 40 years old. It is now very likely that small monoclonal B-cell populations may be identified incidentally during investigations for lymphoma, and so it is increasingly necessary to understand the factors that can differentiate an incidental finding of MBL from bone marrow or tissue involvement with a low level of lymphoma.

In CLL there is relatively even distribution between peripheral blood and bone marrow [START_REF] Rawstron | International standardized approach for flow cytometric residual disease monitoring in chronic lymphocytic leukaemia[END_REF] and the same appears to be true for MBL. In a series of 306 MBL cases from the Mayo Clinic, 46 underwent a bone marrow aspirate and/or biopsy within one year of diagnosis predominantly for staging (n=33), evaluation of cytopenia (n=8).

Most (45/46) showed a nodular and/or interstitial infiltrate. By flow cytometry CLL cells represented a median 10% of leucocytes (range 5-80%). Extensive marrow infiltration (>70% CLL cells) occurred in 3/46 cases, none of which presented with peripheral cytopenias or required any treatment with up to 5 years follow-up [START_REF] Herrick | Monoclonal B-cell lymphocytosis (MBL): A bone marrow study of an indolent form of chronic lymphocytic leukemia (CLL)[END_REF]. In a study from Italy, the median percentage of CLL cells in the bone marrow was 20% (range 3 -65%) with 2/84 showing an extensive bone marrow infiltration [START_REF] Rossi | The prognosis of clinical monoclonal B cell lymphocytosis differs from prognosis of Rai 0 chronic lymphocytic leukaemia and is recapitulated by biological risk factors[END_REF]. In the majority of cases the percentage of CLL cells in the bone marrow will be similar to that in the peripheral blood, therefore one would expect 10-30% CLL cells in the bone marrow in the bone marrow of an indivudal diagnosed with CLL-type MBL. Under most circumstances the presence of up to 5,000 CLL cells/µL in the peripheral blood and/or 30% CLL cells in the bone marrow is not sufficient to affect haematopoietic activity. Unless there is an immune-mediated cytopenia, it is unlikely that CLL-type were detected incidentally [START_REF] Winstanley | Incidental findings in pelvic lymph nodes at radical prostatectomy[END_REF]. In a second study of 4,831 prostate specimens [START_REF] Abrisqueta | Improving survival in patients with chronic lymphocytic leukemia (1980-2008): the Hospital Clinic of Barcelona experience[END_REF]405 biopsies, 266 transurethral resections, 1,160 prostatectomies) there were 18 incidental lymphomas (0.37%) of which 10 involved pelvic nodes only and 8 were primary prostatic lymphoma; 13/18 were CLL, 3 marginal zone lymphoma, 1 mantle cell lymphoma and 1 unclassifiable. The authors note that the lymphomas were not readily apparent in most cases by histologic examination, and may be confused with chronic prostatitis when the prostate was involved or with reactive lymphoid hyperplasia when pelvic nodes were involved [START_REF] Chu | Incidental and concurrent malignant lymphomas discovered at the time of prostatectomy and prostate biopsy: a study of 29 cases[END_REF]. In a follow-up study of 13 of the cases, most (8/13) showed no evidence of systemic dissemination to peripheral blood or bone marrow after a median 43 weeks follow-up [START_REF] He | Clinical outcome of incidental pelvic node malignant B-cell lymphomas discovered at the time of radical prostatectomy[END_REF]. Incidental findings of lymphoma have also been reported in 2/107 (1.9%) hepatic resection undertaken after abnormalities also detected incidentally on ultrasound, CT or MRI [START_REF] Liu | Hepatic resection for incidentaloma[END_REF] and in 5/320

MBL
(1.6%) sentinel lymph node biopsies for early stage breast cancer, of which only 1/5 required treatment for disseminated disease [START_REF] Fox | Incidental lymphoma during sentinel lymph node biopsy for breast cancer[END_REF].

In Incidental detection of tissue involvement with a low level of monoclonal B-cells and some preservation of normal architecture is a relatively rare occurrence in terms of the total number of biopsies investigated, but may represent up to 10% of cases diagnosed with a specific lymphoma. Such samples are of scientific benefit in providing a tool to investigate the early stages of oncogenesis. However, the classification given to individuals is important, particularly in situations where there is a very low probability of developing systemic disease or requiring treatment, because there may be significant psychological and financial implications of a diagnosis of lymphoma [START_REF] Shanafelt | Quality of life in chronic lymphocytic leukemia: an international survey of 1482 patients[END_REF][START_REF] Marti | Diagnostic criteria for monoclonal B-cell lymphocytosis[END_REF].

Occult B-lymphoproliferative disorders

There is a now a significant amount of evidence to indicate that low levels of

  and organomegaly, or ii) associated autoimmune/infectious disease, or iii) B-lymphocyte count >5 • 109/l, or iv) any other feature diagnostic of a B-lymphoproliferative disorder. However, a paraprotein may be present or associated with MBL and should be evaluated independently. 4) Sub-classification: i) CD5+23+: this is the major subcategory and corresponds to a CLL immunophenotype (Cheson et al, 1996). ii) CD5+23-: correlate moderate level of CD20 and CD79b expression with atypical CLL. iii) CD5-: corresponds to non-CLL lymphoproliferative disorder

  prevalence of 0.14% overall and 0.9% in individuals over 60 years of age[START_REF] Rachel | Monoclonal B-cell lymphocytosis in blood donors[END_REF].

  region[START_REF] Rawstron | Monoclonal B-cell lymphocytosis and chronic lymphocytic leukemia[END_REF], CD5-negative MBL was identified in 73 cases and CD5+23-MBL in 44 cases . The overall survival for these disorders, (unpublished data shown in Figure1), is considerably worse for CD5-ngeative MBL and CD5+CD23-MBL and this difference is largely due to the fact that CD5-negative and CD5+CD23-MBL are more likely to have significant tissue or bone marrow infiltration. In cases initially diagnosed through peripheral blood investigation, fewer than 5% of CLL-type MBL will have significant bone marrow or tissue disease detected within one year of diagnosis compared to 20-25% for CD5-negative and CD5+CD23-MBL.

Figure 1 :

 1 Figure1: Overall survival for MBL according to phenotype. Five year survival for CLL-phenotype MBL was 69%, for CD5-negative MBL was 57%, and for CD5+CD23-MBL was 45%. The data does not take into account subsequent diagnosis of lymphoma and the difference in survival is most likely to reflect more extensive underlying disease in cases with a non-CLL phenotype.

  with clinical follow-up 3/13 cases developed follicular lymphoma at 12, 13, and 72 months from diagnosis whilst 10/13 have shown no other evidence of follicular lymphoma[START_REF] Cong | In situ localization of follicular lymphoma: description and analysis by laser capture microdissection[END_REF]. Monotypic B-cell expansions do occur in reactive nodes and in these situations the expanded population usually has the phenotype of normal germinal centre B-cell with very strong CD19, CD20 and CD38 expression and moderate expression of CD10 without co-expression of CD23 expression (unpublished observation).

  oligoclonal or monoclonal B-cell populations with the characteristics of Blymphoproliferative disorders are a frequent finding, both in the general population and as an incidental finding in hospital investigations. Most studies have focused on CLL-type MBL, largely because CLL is one of the most common haematological malignancies and the cells of interest can be obtained from peripheral blood. Scientific studies comparing MBL and CLL are now starting to allow discrimination of factors responsible for oncogenesis and disease progression. For patients, the of CLL-type MBL provides some protection from the anxiety and potential financial issues associated with a diagnosis of leukaemia/lymphoma. It is clear that a similar situation exists for most B-cell malignancies and it is important that similar consideration is given to scientific investigations as well as to the diagnostic implications of these premalignant B-lymphoproliferative disorders. cells among B-cell subsets in healthy individuals. Br. J. Haematol. 2007 Aug;138(3):349-353.

Table 1 : Originally proposed diagnostic criteria for Monoclonal B-cell Lymphocytosis

 1

[START_REF] Marti | Diagnostic criteria for monoclonal B-cell lymphocytosis[END_REF]

Table 2 : Reported prevalence of CLL-type and CD5-negative MBL in population studies.

 2

	Study Group		Flow Cytometry	MBL Prevalence
	Source	Median age (range)	n	No. of colors	CLL-specific	Events x 10³	Overall	CLL-type	CD5-negative
	U.S. residential population	53 (40-78)	1926	2	No	N.S.	0.57	0.28	0.28
	U.K. hospital outpatients	57 (40-90)	910	4	Yes	200	4.5	3.5	1
	Italy primary care	74 (65-98)	500	4	Yes	200	6.9	5.5	1.4
	U.K. hospital outpatients	74 (60-80)	1520	4	No	200	6.9	5.1	1.8
	Italy residential population	55 (18-102)	1725	5	Yes	500	7.4	6.3	1.2
	Spain primary care	62 (40-97)	608	8	Yes	5000	14.3	12.0	2.3

The prevalence of CD5-negative MBL is approximately 1-2% independent of assay sensitivity. MBL with a germinal centre phenotype is not reported in otherwise healthy individuals

  Although the reported prevalence of CLL-type MBL has increased significantly over time, there has been a more modest change in the proportion of individuals in the population reported to have a CD5-negative MBL. Even in studies analysing very large numbers of cells and using 10 parameter flow cytometry, the prevalence of CD5-negative MBL is 2.3% compared to 12% for CLL-type MBL[START_REF] Nieto | Increased frequency (12%) of circulating chronic lymphocytic leukemialike B-cell clones in healthy subjects using a highly sensitive multicolor flow cytometry approach[END_REF] and in keeping with this most studies report a prevalence for CD5-negative MBL that is approximately 5-five lower than for CLL-type MBL (see Table2). Furthermore,

	individuals (16/327) having more than 0.01% cells with a t(14;18) compared to 94%
	(7/108) of people with newly diagnosed follicular lymphoma. The median number of
	circulating cells with a translocation was 5.8 per million mononuclear cells (range
	0.5-1079) in healthy individuals compared to 9200 per million mononuclear cells
	(range 7-1,000,000) in individuals with a new diagnosis of follicular lymphoma (24).
	In spite of this difference, if the cells with a t(14;18) had a phenotype consistent with
	follicular lymphoma it would be expected that high sensitivity flow cytometry studies
	would detect CD10+ monoclonal B-cells in at least 2% of individuals. It is possible
	that current flow cytometry approaches are unsuitable for detection of very low levels
	of follicular lymphoma in the peripheral blood, but this seems unlikely as circulating
	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	of the general population, with sequence analysis demonstrating that individuals with
	a t(14;18) translocation often harboured several (2-5) unrelated B-cell clones with the
	abnormality (20,21). The translocation is detected in a much higher proportion within
	the IgM+ memory B-cell subset compared to isotype-switched memory B-cells and
	naïve B-cells (22,23). There is a strong association between age and the prevalence of
	t(14;18) with the translocation not detected in individuals under ten years old, rising

studies including assessment of CD10 expression on monoclonal B-cells report no cases of CD5-negative MBL co-expressing CD10

[START_REF] Nieto | Increased frequency (12%) of circulating chronic lymphocytic leukemialike B-cell clones in healthy subjects using a highly sensitive multicolor flow cytometry approach[END_REF][START_REF] Ghia | Monoclonal CD5+ and CD5-B-lymphocyte expansions are frequent in the peripheral blood of the elderly[END_REF]

and most CD5-negative MBL have a phenotype more consistent with marginal zone lymphoma

[START_REF] Nieto | Non-CLL-like monoclonal B-cell lymphocytosis in the general population: Prevalence and phenotypic/genetic characteristics[END_REF]

. This is surprising given the relatively high prevalence of detecting the t(14;18) translocation in the peripheral blood of healthy individuals. The t(14;18) translocation is the hallmark genetic abnormality in follicular lymphoma with rearrangement of the BCL2 gene from 18q21 to the immunoglobulin heavy chain gene on 14q32. BCL2 is an anti-apoptotic molecule that is strongly expressed at most stages of B-cell differentiation but is not present in normal germinal centre B-cells. The t(14;18) translocation is thought to cause over-expression of BCL2 which permits aberrant survival of affected cells at the germinal centre stage. Alternative BCL2 translocations to immunoglobulin light chain genes have also been reported

[START_REF] Swerdlow | WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues[END_REF]

. The translocation is also detected in the B-cells of a large proportion of the more than half to a maximum of 66% of individuals aged 40-49 years, and then remaining stable at 50-60% prevelance in individuals aged over 50 years old. In population studies the numbers of cells with the translocation is generally low with fewer than 5% of healthy cells are readily detectable with available techniques in clinical disease

[START_REF] Morice | Predictive value of blood and bone marrow flow cytometry in Bcell lymphoma classification: comparative analysis of flow cytometry and tissue biopsy in 252 patients[END_REF][START_REF] Sánchez | Incidence of phenotypic aberrations in a series of 467 patients with B chronic lymphoproliferative disorders: basis for the design of specific four-color stainings to be used for minimal residual disease investigation[END_REF]

which suggests that the circulating cells with a t(14;18) translocation identified by PCR studies in healthy individuals do not have a phenotype consistent with follicular lymphoma. It also indicates that the detection of monoclonal B-cells with a follicular lymphoma phenotype is likely to indicate clinical disease.

  MBL cases from hospital clinic series[START_REF] Rawstron | Monoclonal B-cell lymphocytosis and chronic lymphocytic leukemia[END_REF][START_REF] Shanafelt | Natural History of Individuals With Clinically Recognized Monoclonal B-Cell Lymphocytosis Compared With Patients With Rai 0 Chronic Lymphocytic Leukemia[END_REF] but different usage is seen in individuals with a very low (<10 cells per µL) CLL cell count[START_REF] Nieto | Increased frequency (12%) of circulating chronic lymphocytic leukemialike B-cell clones in healthy subjects using a highly sensitive multicolor flow cytometry approach[END_REF][START_REF] Dagklis | The immunoglobulin gene repertoire of low-count chronic lymphocytic leukemia (CLL)-like monoclonal B lymphocytosis is different from CLL: diagnostic implications for clinical monitoring[END_REF]. Together the data indicate that there is a common genetic background and set of chromosomal abnormalities that lead to the development of both CLL-type MBL and CLL but significant expansion of CLL cells only occurs in clones with specific immunoglobulin genes. Similar data is not available for other types of MBL. As noted above, MBL with a germinal centre phenotype has not been reported and whilst CD5+CD23-MBL occurs in clinic series it is very rare in population studies. CD5-negative MBL typically has a phenotype consistent with marginal zone lymphoma or lymphoplasmacytic lymphoma and although clonal genetic abnormalies are detectable there are limited data available on underlying genetic factors associated with susceptibility or oncogenesis

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. The immunoglobulin gene usage is restricted in CLL and many cases use closely homologous (stereotyped) complementarity determining regions

[START_REF] Stamatopoulos | Over 20% of patients with chronic lymphocytic leukemia carry stereotyped receptors: Pathogenetic implications and clinical correlations[END_REF]

. Similar immunoglobulin gene usage is seen in CLL-type

Incidental detection of B-lymphoproliferative disorders in tissue biopsies

  will have any effect on other haematological parameters. Although there is little In the germinal centre DLBL, none of the MBL cases had a CLL-phenotype whereas 2/11 were CLL-phenotype in the ABC DLBL. In all cases the MBL cells represented less than 5% of total bone marrow leucocytes. None of the MBL cases expressed CD10, even if the DLBL was CD10+. It was difficult to determine whether there was a clonal relationship between the MBL and DLBL because there are often different markers used for immunohistochemistry and flow cytometry and amplifiable DNA could only be extracted in one of the cases.

	published data, it appears that cytopenia in individuals with CLL-type MBL is usually
	caused by unrelated abnormalities such as dysplasia, anaemia of chronic disease or
	metastatic carcinoma.
	Flow cytometry studies in the bone marrow of individuals with Diffuse Large B-cell Incidental finding of lymphoma in tissue biopsies varies according to the site and
	Lymphoma (DLBL) demonstrate that MBL is detectable in up to 30% of cases reason for investigation. Approximately 1 in 300 surgical prostate investigations
	depending on sub-category. MBL was relatively infrequent (3.7%, 3/80) in cases with result in an incidental detection of lymphoma according to two independent studies.
	a germinal centre phenotype but was more common (28.2%, 11/39) in ABC-type In a series from the UK reviewing 854 lymph node biopsies removed during radical
	F o r P e e r F o r P e e r DLBL. Of 8228 bone marrow aspirate with paired trephine biopsy samples investigated prostatectomy, 3/854 (0.35%) cases of lymphoma (2 CLL, 1 follicular lymphoma)
	R e v i e between 2006 and 2009 at the HMDS laboratory, flow cytometry revealed a R monoclonal B-cell population of in 151/8228 (1.8%) cases which was not evident by e morphology on the trephine biopsy or could not be adequately characterised (unpublished observation). However, in the vast majority of cases the flow cytometry data could either confirm absence of disease, provide evidence to support the presence v i e
	w of disease or provide complete characterisation of the neoplastic cells present. In bone marrow aspirate and trephine biopsies taken to assess response to treatment for CLL, w
	over 30% will have residual disease detectable by flow cytometry but with no disease
	detectable on the trephine biopsy (7). Therefore flow cytometry investigations have
	significant power to improve the detection of disease in bone marrow aspirate
	samples. However, some care must be taken to interpret low levels of bone marrow
	involvement. In particular, incidental finding of CLL-type MBL and CD5-negative
	MBL may be present in 1-2% of samples. In a staging bone marrow from a patient
	with lymphoma, the detection of a low level (e.g. <1% of leucocytes &/or <50% of
	total B-cells) monoclonal B-cell population does not demonstrate bone marrow
	involvement with lymphoma unless there is additional evidence of clonal identity.

  our institution, review of lymph node biopsies with additional flow cytometry (3.5%) of cases diagnosed as follicular lymphoma (unpublished observation). In other series, the proportion of cases with at least on conserved reactive follicle was reported to be as 10.6% of total with the majority of having limited stage (I/II) disease[START_REF] Adam | Presence of preserved reactive germinal centers in follicular lymphoma is a strong histopathologic indicator of limited disease stage[END_REF]. In a series of 23 cases with focal BCL2+ follicles but otherwise predominantly reactive follicles, 5/23 had evidence of follicular lymphoma at another site but of 13 cases

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	available on freshly disaggregated cells demonstrated only partial nodal involvement
	with maintenance of normal follicles and a mixture of polyclonal and monoclonal B-
	cells in 4/50 (8%) cases diagnosed as CLL. There was no evidence of a lymphocytosis
	and if a solitary node had not been excised the diagnosis would have been MBL rather
	than CLL. These cases highlight the fact that there is probably a significant number of
	individuals diagnosed with lymphoma who have only minimal involvement, possibly
	in isolated nodes. Low level of nodal involvement also occurs for other disorders, for
	example in 3/40 (7.5%) cases diagnosed as marginal zone lymphoma and 10/286

Published on behalf of the British Division of the International Academy of Pathology Histopathology


teaching and research institutions in France or abroad, or from public or private research centers.