research paper

Monoclonal B-cell lymphocytosis in first-degree relatives of patients with sporadic (non-familial) chronic lymphocytic leukaemia

Daniel M. Matos,1 Sebastia˜o J. Ismael,1 Summary Carlos A. Scrideli,2 Fa´bio M. de Oliveira,1 Eduardo M. Rego1 and Roberto P. Although biological similarities have been described among monoclonal Falca˜o1 B-cell lymphocytosis (MBL) and chronic lymphocytic leukaemia (CLL), the Departments of 1Clinical Medicine and Centre for relationships between these two conditions are not fully understood, and new Research on Cell-Based Therapy, and 2Paediatrics, epidemiological studies in different populations and different countries School of Medicine of Ribeira˜o Preto, University of continue to be reported. Here, we investigated 167 first-degree relatives from Sa˜o Paulo, Ribeira˜o Preto-SP, Brasil 42 families of patients with non-familial (sporadic) CLL, using four-colour flow cytometry. MBL was found in seven of 167 subjects (4Æ1%). Monoclonality was detected in all cases either by light-chain restriction or by polymerase chain reaction. Fluourescence in situ hybridization did not show any chromosomal abnormality. The prevalence of MBL according to age was 0 (0/54) in individuals aged less than 40 years, 2Æ5% (2/81) between Received 26 May 2009; accepted for publication 9 July 2009 40 and 60 years, and 15Æ6% (5/32) in individuals over 60 years. The Correspondence: Roberto Passetto Falca˜o, MD, prevalence of MBL cases in individuals over 60 years was similar to that PhD, Department of Clinical Medicine, School found in familial CLL relatives at the same age group. This suggests that in of Medicine of Ribeira˜o Preto, University of Sa˜o older first-degree relatives of patients with sporadic CLL, the risk of MBL Paulo (USP), Avenida Bandeirantes, 3900, detection is as high as in older first-degree relatives from CLL families, which 14049-900 Ribeira˜o Preto-SP, Brasil. could render these individuals belonging to ‘sporadic CLL families’ as E-mail: [email protected] susceptible as individuals from ‘familial CLL’ to the development of clinical Potential conflicts of interest: none reported. CLL. This work was supported by a grant from FAPESP (05/59209-0 and 07/52462-7) and Keywords: monoclonal B-cell lymphocytosis, chronic lymphocytic leukae- CNPq (472487/2006). mia, flow cytometry, B cells, first-degree relatives.

Although chronic lymphocytic leukaemia (CLL) represents one organomegaly on physical examination (Marti et al, 2005). of the most frequent haematological malignancies around the The biological proximity between CLL and MBL occurs at world, accounting for 22–30% of all leukaemias (Redaelli et al, various levels: immunophenotypically, MBL cells express the 2004) its aetiology remains largely unknown. In contrast with surface CD5, CD23 and CD20dim, which is a typical other forms of leukaemia, the association of CLL with ionizing finding of CLL cells. Moreover, expression profile radiation, chemical exposure and lifestyle factors is only weak shows that CLL and MBL are identical even when those (Linet et al, 2007). On the other hand, there is a large amount diagnostic markers are excluded. Finally, the proportion of of evidence, based on epidemiological and family studies, that CD5+CD23+ MBL cases that have a 13q14 deletion or trisomy points to an inherited predisposition and, in fact, CLL exhibits 12 is similar to that detected in CLL (Rawstron et al, 2007). one of the strongest familial predisposition (Caporaso et al, Nevertheless, the clinical relationship between MBL and CLL 2007). is not fully understood. Thus, Landgren et al (2009) conducted Recently, the International Familial CLL Consortium a prospective study where 44 of 45 CLL cases diagnosed in defined monoclonal B-cell lymphocytosis (MBL) as the subjects who participated on the Prostate, Lung, Colorectal presence of a clonal B-cell population in the context of an and Ovarian Screening Trial were preceded by MBL, absolute B-cell count <5 · 109 cells/l, no history of auto- which suggested MBL as a precursor state. On the other hand, immune disease and no evidence of lymphadenopathy and Dagklis et al (2009), at a different level of investigation,

First published online 25 August 2009 ª 2009 Blackwell Publishing Ltd, British Journal of Haematology, 147, 339–346 doi:10.1111/j.1365-2141.2009.07861.x D. M. Matos et al reported that MBL and CLL are strikingly distinct with regard to the use of IGHV in an Italian population, which hints that MBL does not represent necessarily a pre-leukaemic condition. Moreover, Shim et al (2007) reported a progression to CLL in only one of eleven individuals with MBL. Fung et al (2007) followed 26 individuals with MBL, and with a median follow-up of 30 months, no case underwent transformation to CLL. Rawstron et al (2008) detected MBL cases in two distinct cohorts of patients: one consisting of normal subjects with normal blood counts, and the other of patients with lymphocytosis. Anony- mous samples were used for studies of subjects with a normal blood count, but, unfortunately, no follow-up data of this group is available. In the second cohort, the rate of progression to CLL Fig 1. Epidemiological characteristics of CLL first-degree relatives. was 1Æ1% per year. In all three studies, MBL cases were identified from the general population and, at present, there are only three Definition of monoclonal B-cell lymphocytosis reports of MBL incidence in the setting of familial CLL, characterized by the presence of two or more individuals with We utilized the recently defined diagnostic criteria for MBL the diagnosis of CLL inside the same family (Marti et al, 2003; (Marti et al, 2005): (i) detection of a disease-specific immu- Rawstron et al, 2002a; de Tute et al, 2006). nophenotype or an overall kappa/lambda ratio >3:1 or <0Æ3:1, Thus, since the clinical relationship between CLL and MBL (ii) stable monoclonal B-cell population over a 3-month in the familial and sporadic CLL remains unsettled period, (iii) absence of lymphadenophaty, organomegaly, (Marti et al, 2007) new epidemiological studies are important autoimmune or infections diseases, and B-lymphocyte counts to better ascertain the biological link among these two <5 · 109/l. conditions, mainly in the sporadic (non-familial) CLL popu- Monoclonal B-cell lymphocytosis was classified as CLL-like lation setting, where, to our knowledge, there are no published (CD5+, CD20dim, CD23+) or atypical CLL (CD5+, CD20bright, ) studies addressing the prevalence, biological characteristics and CD23+/ ). evolution of MBL. Flow cytometry Patients, materials and methods Approximately 10 ml of peripheral blood were collected in EDTA. Mononuclear cells were isolated from samples by Fycoll Patients and study design Hypaque (Sigma-Aldrich, St Louis, MO, USA) density gradient This transversal, descriptive study was performed in first- centrifugation. The final concentration was adjusted to degree relatives of patients with sporadic (non-familial) CLL. A 1Æ0 · 106 cells per tube. The initial flow cytometry panel total of 171 individuals were assessed, derived from 43 families. consisted of three tubes: (i) CD20 [fluorescein isothiocyanate However, two CLL cases were detected from the same family, (FITC)], CD79b [phycoerythrin (PE)], CD19 [PE-cyanin 5 therefore these cases were classified as familial CLL. A group of (Cy5)], CD5 [allophycocyanin (APC)]; (ii) anti-j (FITC), four individuals studied pertaining to this family (the CLL anti-k (PE), CD19 (PE-Cy5), CD5 (APC); (iii) polyclonal anti- patient and three healthy sisters) was excluded from the study. j (FITC), polyclonal anti-k (PE), CD19 (PE-Cy5), CD5 Thus, a total of 167 subjects were included, derived from 42 (APC). All monoclonal antibodies were purchased from families. The distribution by age and sex is shown in Fig 1. The Becton Dickinson (San Jose, CA, USA), except polyclonal heredograms of all families were built (data not shown). The anti-j and anti-k, which were purchased from Dako families were randomly selected and all patients with CLL have (Carpinteria, CA, USA). All samples were analysed using a been followed in the University Hospital, School of Medicine FACSCalibur flow cytometer (Becton Dickinson) equipped of Ribeirao Preto, University of Sao Paulo. with an argon ion laser and a second red diode laser. Prior to The diagnosis of CLL was based on morphology and a score the acquisition of each individual sample, we performed four- of four or five points using the Matutes immunophenotypic colour compensation by using mononuclear cells previously analysis system (Matutes et al, 1994) or, alternatively, on a marked with a FITC, PE, PE-Cy5 and APC, each in separate biopsy showing a histopathological diagnosis of small lym- tubes. A total of 300 000 events per tube were acquired. The phocytic . Cell Quest software (Becton Dickinson) was used for data The study was approved by the University Hospital Ethics acquisition and analysis. Committee, and individuals gave their written informed We used the sequential gating strategy as previously consent prior to entering the study in accordance with the described by Rawstron et al (2001). The objective was to Declaration of Helsinki. identify the population of CD19+/CD5+ B-lymphocytes and

340 ª 2009 Blackwell Publishing Ltd, British Journal of Haematology, 147, 339–346 Monoclonal B-cell Lymphocytosis in Sporadic CLL exclude debris and unspecific binding, by the delimitation of considered as positive, except for CD38, in which case the cut- three regions: (R1) high CD19 expression and low internal off value was of 34%, based on a recent publication (Rassenti complexity [side scatter (SSC)]; (R2) low cellular size [forward et al, 2008). Moreover, to exclude monocytes, T-lymphocytes scatter (FSC)] and low internal complexity; and (R3) positivity and normal B-lymphocytes, the analysis was restricted to for the CD5 in cells CD19+. Subsequently, abnormal cells with CD5+/CD19+/CD20dim B-cells. CLL phenotype were identified by the combination of weak expression of CD20 and CD79b (gates R4, R5 and R6), as Immunomagnetic B-cell sorting shown in Fig 2. A disease-specific immunophenotype was considered when a minimum of 100 events occurred simul- B-lymphocytes from the peripheral blood were isolated by taneously in regions R4, R5 and R6. The expression of kappa magnetic-activated cell sorting (MACS; Miltenyi Biotec, Berg- and lambda chains of immunoglobulin was analysed in the isch-Gladbach, Germany) following the manufacturer specifi- B-lymphocyte CD5+ population with the purpose of detect cations. Briefly, mononuclear cells previously separated by light-chain restriction (j/k > 3/1 or j/k <0Æ3/1). Fycoll Hypaque density gradient centrifugation were incubated To better characterize the MBL cases, the immunopheno- with an anti-human CD19 monoclonal antibody conjugated type panel was extended using the following antibodies: with microbeads (Miltenyi Biotec). The cell suspension was CD11a, CD23, CD38, CD49c, CD49d, CD54 and FMC7. A passed through a LS column (MACS Separation Columns; cut-off point of 30% of cells expressing a given marker was Miltenyi Biotec) previously attached to the SuperMACS device 4 PCZ 2.003 PCZ 2.0034 PCZ 2.003 10 R1 10 R1 R2 R3 R3 3 3 10 10 2 2 10 10 SSC-H 1 1 CD19 PE-Cy5 R2 CD19 PE-Cy5 10 10 0 0 0 200 400 600 800 1000 10 0 200 400 600 800 1000 0 200 400 600 800 1000 10 0 1 2 3 4 SSC-H FSC-H 10 10 10 10 10 CD5 APC 4 PCZ 2.003 4 PCZ 2.003

4 PCZ 2.003 10

10 R5 R4 R5 10 R6 3 3

3 R6 10 R4 10 10 2 2 2 10 10 10 CD5 APC CD5 APC 1 1 CD79b PE 1 10 10 10 0 0 0 10 10

100 101 102 103 104 100 101 102 103 104 10 100 101 102 103 104 CD20 FITC CD79b PE CD20 FITC

4 PCZ 2.006 4 PCZ 2.006 10 10 AB 3 3 10 10 2 2 10 10 CD5 APC CD5 APC 1 1 10 10 0 0 10 1010 0 101 102 103 104 100 101 102 103 104 LAMBDA RPE DK KAPPA FITC DK

Fig 2. Monoclonal B-cell lymphocytosis in a first-degree relative of a patient with sporadic CLL. (R1) High CD19 expression and low side scatter. (R2) Low forward scatter and low side scatter. (R3) Positivity for the CD5 in cells CD19+. (R4), (R5) and (R6) Abnormal B-cells are gated, based on weak CD20 and CD79b expression. A and B show a monoclonal B-cell population, CD5+/Kappa+.

ª 2009 Blackwell Publishing Ltd, British Journal of Haematology, 147, 339–346 341 D. M. Matos et al for positive selection of labelled cells. The purified CD19+ two cases each), of a total of 167 subjects studied (4Æ1%). One population was used for Polymerase Chain Reaction (PCR) familial CLL was detected and excluded from this analysis. The and fluorescence in situ hybridization (FISH) experiments. prevalence according to age was 0 (0/54) in individuals aged less than 40 years, 2Æ5% (2/81) in individuals aged between 40 Polymerase chain reaction for immunoglobulin heavy-chain and 60 years, and 15Æ6% (5/32) in individuals aged over (IGHV) and T-cell (TCR) genes. DNA was extracted 60 years. Six cases were monoclonal B-cell lymphocytosis CLL- from purified CD19+ B-lymphocytes using the Wizard like, and just one presented as atypical CLL. The median Genomic DNA Purification kit (Promega, Madison, WI, percentage of abnormal cells was 7Æ5% (range, 4–20%) of all USA), according to manufacturer instructions. DNA was B-lymphocytes. Table I shows the characteristics of MBL cases. quantified by spectrophotometry and tested for quality by In Individual P1, who presented a MBL with the atypical standard PCR amplification for HBB. DNA samples from the CLL phenotype, the immunophenotype panel was extended cells collected were amplified by PCR with consensus primers using a different strategy, where the k+/CD5+/CD19+/CD20+ flanking the CDR-3 region of IGHV (FR3A, LJH, VLJH), TRG B-cells were gated, because the abnormal clone had a bright (TRGV1-4 and TRGJ1-2/TRGJP/TRGJP1–2 families), CD20 expression. incomplete TRD (TRDV2/TRDD3) and the PCR product was In Individual P6, flow cytometric analysis was repeated submitted to homo/heteroduplex analysis as previously 9 days after (and not 3 months later, as with the other described (Scrideli et al, 2004a,b) and presented a sensitivity individuals) because the subject refused to be followed-up in ) ) ranged from 10 2 to 10 3. Clonality was characterized by the the University Hospital. However, the second analysis presence of a band of the expected size in the homo/ undoubtedly confirmed the presence and the stability of the heteroduplex analysis on 12% polyacrylamide gel. The MBL clone. Moreover, repeat flow cytometry is not essential expected fragment sizes are: 80–120 bp for CDR 3 (IGHV), for research applications (Marti et al, 2005), provided that the 80–100 bp for TRD, 170–210 bp for TRG. B-cell clone was clearly demonstrated by either an abnormal kappa/lambda ratio (in this case: j/k =4Æ35:1) and, concom- Fluorescence in situ hybridization (FISH). Interphase itantly, by the presence of an immunophenotypically abnormal fluorescent in situ hybridization analysis (FISH) was B-cell population (in this case: CD5+/CD20+dim/CD79b+dim B performed in isolated CD19+ B-lymphocytes from peripheral cells). blood using Vysis LSI p53/LSI ATM and LSI D13S319/LSI With a median follow-up of 23 months (range 10– 13q34/CEP 12 Multi-colour probe set (Downers Grove, IL, 28 months), none of the MBL cases progressed to CLL or USA). The cells were fixed in 1/1 (vol/vol) methanol/acetic and other B-cell chronic lymphoprolipherative disorder. stored at )20C. Fixed cells were prepared and hybridized according to the manufacturer’s instructions. Briefly, slides Clonal rearrangements detection by PCR containing both the cell DNA and probe DNA were denaturated at 75C for 5 min and immediately hybridized In six out of the seven MBL cases, CD19+ B-cells were isolated (overnight at 37C), in a humid chamber. Then, slides were and DNA was analysed by PCR. The percentage of purified sequentially washed (5 min at 45C) in 50% formamide/2· CD19+ cells in Individuals P1, P2, P3, P4, P5 and P7 was 85%, sodium chloride citrate buffer and phosphate-buffered saline 85%, 75%, 70%, 72% and 92%, respectively. with 1% Nonidet P-40 (vol/vol), and counterstained with IGHV clonal gene rearrangements were detected in four 20 ll of DAPI (4¢-6-diamidino-2-phenylindole; Sigma). The individuals (P1, P3, P4 and P5) (Fig 3A). In two subjects, hybridization spots were evaluated using a fluorescence Individuals P3 and P5, we also detected TRG clonal microscope (Axioskop 40; Carl Zeiss, Jena, Germany). For rearrangements in the MBL population (Fig 3B). These each sample, the number of hybridization spots per nuclei was individuals presented a clear immunophenotypically abnormal ) counted in 400 nuclei. Only signals with a similar size, CD5+/CD20+dim/CD23+/CD79b+dim/FMC7 B-cell popu- ) intensity and shape were counted. The cut-off values were lation, which contrasts with the CD5+/CD20+bright/CD23 / based on FISH studies of normal control peripheral blood CD79b+bright/FMC7+ normal peripheral blood B-cells (Marti cells. The upper limits of normal (mean + 3 standard et al, 2003). These abnormal populations represented 5Æ1% deviations) were set at 3%, 4%, 6% and 3%, for trisomy 12, and 4% of all B-lymphocytes in Individuals P5 and P3, del(13q), del(11q23Æ3) and del(17p13Æ1), respectively. FISH respectively. analyses were undertaken by a single observer. Fluorescence in situ hybridization (FISH) Results FISH panels were applied to reliably detect genetic abnormal- ities of proven clinical significance. However, FISH analysis Prevalence of monoclonal B-cell lymphocytosis performed on purified CD19+ B-cells from six of seven subjects Monoclonal B-cell lymphocytosis was detected in seven-first- displaying MBL did not show any chromosomal abnormality degree relatives, derived from five families (two families had (Table II).

342 ª 2009 Blackwell Publishing Ltd, British Journal of Haematology, 147, 339–346 Monoclonal B-cell Lymphocytosis in Sporadic CLL

(A) 1 2 3 4 5 6 7 8 9 10 11 PCR Monoclonal Negative Monoclonal Monoclonal Monoclonal Negative 100 bp + ) ) ) ) ) /FMC7 /FMC7 + + /FMC7 /FMC7 /FMC7 /FMC7 + + + + M P P P P Cpb Cbm C+ C– P P 1 247 3 5 /CD54 /CD54 + + /CD54 /CD54 /CD54 /CD54 ) ) ) + (B) 12345 /CD49d /CD49d ) + /CD49d /CD49d /CD49d /CD49d + + + +

/CD49c /CD49c 200 bp ) ) /CD49c /CD49c /CD49c /CD49c + ) ) ) /CD38 /CD38 + + /CD38 /CD38 /CD38 /CD38 + + + + MPC– C+ P /CD23 /CD23

/CD23 /CD23 /CD23 /CD23 5 3

+bright +bright +dim +dim +dim +dim Fig 3. PCR homo/heteroduplex analysis. (A) IGHV rearrangements: line 1 (M) molecular weight marker; line 2 (P1), line 4 (P4), line 10 (P3) and line 11 (P5): individuals with monoclonal IGHV rearrange- ments; line 3 (P2) and line 5 (P7): individuals with polyclonal IGHV rearrangements; line 6 (Cpb), line 7 (Cbm), polyclonal controls from ratio Extended phenotyping k 08:125:1 CD11a 28:1 CD11a 12:1 CD11a 35:1 CD11a 35:1 CD11a 94:1 Not performed CD11a Not performed :

Æ Æ Æ Æ Æ Æ Æ healthy donor samples of peripheral blood and bone marrow respec- j 0 0 1 1 1 4 4 tively; line 8 (C+) monoclonal positive control and line 9 (C)) negative control (no DNA). (B) TRG gene rearrangements: line 1 (M)

+dim molecular weight marker; line 2 (C)) negative control (no DNA); line +dim +dim +dim +dim +dim +dim 3 (P5) and line 5 (P3), monoclonal gene rearrangements; line 4 (C+), monoclonal positive control. /CD79b /CD79b /CD79b /CD79b /CD79b /CD79b /CD79b

+bright +dim +dim +dim +dim +dim +dim Table II. Chromosomal abnormalities of MBL cases. /CD20 /CD20 /CD20 /CD20 /CD20 /CD20 /CD20

+ + + + + + + Chromosomal abnormalities (%)

MBL imunophenotype Individual Trisomy 12 Del(13q) Del(11q) Del(17p) 6 CD5 8 CD5 8 CD5 5 CD5 1 CD5 Æ Æ Æ Æ Æ P1 2Æ02Æ03Æ02Æ0

% MBL* P2 3Æ03Æ02Æ02Æ0 P3 2Æ01Æ52Æ02Æ0 P4 3Æ01Æ04Æ03Æ0 /l)

9 P5 3Æ01Æ53Æ02Æ0 10 8 20 CD5 07 25 18 77 385 4 CD5 Æ Æ Æ Æ Æ Æ Æ P6 Not Not Not Not · Lymphocytes ( performed performed performed performed P7 2Æ01Æ52Æ02Æ0

/l) Cut-off (%) 3Æ04Æ06Æ03Æ0 9 10 41 44 11 11 61 51 11 Æ Æ Æ Æ Æ Æ Æ · Leucocytes (

Discussion The present study determined the prevalence ratio of MBL in Age (years)/sex 167 first-degree relatives deriving from 42 families of patients with sporadic CLL. We have found seven cases of MBL: five males and two females. These results are analogous to those found in CLL, which demonstrated that MBL is at least twice Age, sex and laboratorial characteristics of MBL cases. more common in males than in females (Redaelli et al, 2004). In agreement with Dagklis et al (2009), we also detected Also monoclonal for T-cell receptor. Table I. Individual Family P1 1 46/F 5 P7 5 61/F 8 P4P6 4 5 53/M 75/M 8 5 P2P3P5 2 3 75/M 4PCR, 72/M polymerase chain reaction. *Percentage to a 5 total of 62/M B-cells. 4 9 cases of MBL with a normal kappa/lambda ratio. These

ª 2009 Blackwell Publishing Ltd, British Journal of Haematology, 147, 339–346 343 D. M. Matos et al individuals had the lowest percentage of abnormal B-cells donors, which was considerably lower than previous reports, among the MBL cases (Table I). However, monoclonality was probably because the samples were initially analysed only by detected in all of our cases either by light-chain restriction dual-colour flow cytometry. Otherwise, Nieto et al (2009) analysis, PCR, or both. A monoclonal IGHV band was detected found a MBL prevalence of 12% in a Spanish population with by PCR in four of six of our cases, and this is in agreement the use of eight-flow colour flow cytometry approach. It seems with the percentage detected in patients with CLL (Trainor that the less sensitive assays, based only on the kappa/lambda et al, 1990). Moreover, in the study of Rawstron et al (2002b), ratio, hamper the detection of MBL, and, on the other hand, a monoclonal rearrangement was demonstrated in eight out of the more sensitive 6–8-colour flow cytometry, albeit very 12 cases, which is agreement with our results. Monoclonality useful, could not be used on this occasion to compare the could not be demonstrated by PCR in all cases probably due to prevalence of MBL between different populations, provided the unspecific binding of consensus primers on germline that the studies which have been using it are scarce. sequences of polyclonal B-cells found in the background. So, when we consider the four-colour flow cytometry assay, Moreover, as previously reported, we have demonstrated that currently applied globally, the overall prevalence of MBL found the four-colour flow cytometry assay with the use of the in our population (4Æ1%) is similar to that reported for the combination CD20, CD79b, CD19 and CD5 antibodies is general population, which ranges between 3Æ5% and 5Æ5% more sensitive than the kappa/lambda ratio and even than (Ghia et al, 2004; Rawstron et al, 2002b), but is lower than the PCR (Rawstron et al, 2001). prevalence of MBL in healthy individuals belonging to CLL TRG clonal rearrangement was also found in two subjects families, which ranges between 13Æ5% and 18% (Marti et al, from the MBL population. T-cell receptor gene rearrangements 2007; Rawstron et al, 2002a). Even if different populations other than those of the b chain genes (Chen et al, 1987; Dyer, were evaluated, there is good concordance between various 1989; Merup et al, 1994; Norton et al, 1988) are extremely rare studies in different countries (Rawstron, 2004), which almost in CLL (Kerlan-Candon et al, 1998; Perl et al, 1990). As far as completely precludes any bias of ethnical selection and enables we know, the two cases described here are the first published the comparison of our research population with others cases of MBL harbouring clonal rearrangements of a T-cell worldwide. The study of Dagklis et al (2009), performed in a receptor gene, and probably they were detected because we North Italian community, reinforces this statement, as the have usually used a PCR strategy which includes consensus reported prevalence of 7Æ4% of all MBL subtypes, and of 6Æ3% primers to search for TCR clones in all of our MBL cases. when just atypical CLL MBL and CLL-like MBL were We did not detect any cytogenetic abnormalities in six of considered, is similar to that reported by previous studies. seven subjects analysed by FISH. Although chromosomal Chronic lymphocytic leukaemia is mainly a disease of the lesions associated with poor prognosis (17p and 11q deletions) elderly (Redaelli et al, 2004), which indirectly indicates an age- are rare in MBL (Rawstron et al, 2007), recent studies have related premalignant condition. In fact, it has been previously reported that 13q14 deletion and trisomy 12 have been found reported that MBL in normal individuals mimics that of in similar proportions in MBL and in CLL patients (Rawstron clinical CLL, and, in contrast, the proportion of affected et al, 2008). Our negative FISH results could be explained by individuals in familial CLL is high at all ages (Rawstron et al, the low median percentage of the MBL cells (7Æ5%, range 2007). Therefore, it is more suitable to analyse the prevalence 4–20%), which, at least in some cases, could be lower than rates of MBL at specific age groups, which could provide more limit of FISH sensitivity. Anyway, our results do not enable information about the relationship between CLL and MBL meaningful conclusions to be drawn regarding the cytogenetic from the epidemiological point-of-view. profile of MBL in sporadic CLL context. Regarding the analysis of MBL in age groups, we found a With a median follow-up of 23 months, we did not detect prevalence of 15Æ6% (5/32) in individuals over 60 years old, any case that progressed to CLL or other B-cell chronic which was higher than values previously reported for the same lymphoproliferative disorder. Our follow-up results are similar age group in the general population (Ghia et al, 2004; to those of Fung et al (2007). These authors followed 26 Rawstron et al, 2002a), but similar [16Æ7% (3/18)] to the patients with MBL, and with a median of follow-up of prevalence reported by de Tute et al (2006) in familial CLL 30 months, no case underwent transformation to CLL. Raw- subjects over 60 years old (Table III). Thus, the overall stron et al (2008) have reported progression from MBL to prevalence of MBL in first-degree relatives of patients with CLL, but the median follow-up in this study was much longer sporadic CLL is lower than in relatives with familial CLL, but (6Æ7 years) than in the present study. Moreover, it is also our results suggest that the prevalences may be similar in the important to take into consideration that these MBL individ- over 60s. uals were selected from the general population, and, at the The results of the follow-up study of MBL subjects recently present time, it is not known whether MBL from sporadic CLL reported by Rawstron et al (2008) showed that CLL requiring families have a different evolution. treatment developed in individuals with MBL and absolute The calculated prevalence of MBL is rather influenced by the lymphocytosis at the rate of 1Æ1% per year, which suggests that various flow cytometry techniques utilized. Recently, Rachel MBL is a new premalignant condition. Thus, provided that the et al (2007) reported a prevalence of 0Æ14% among blood higher risk of emergence of clinical CLL in relatives of CLL

344 ª 2009 Blackwell Publishing Ltd, British Journal of Haematology, 147, 339–346 Monoclonal B-cell Lymphocytosis in Sporadic CLL

Table III. Prevalence of MBL in three different populations. wrote the paper. R.P.F, designed research, supervised the study and wrote the paper. Age Normal relatives Normal relatives group General from CLL from sporadic (years) population* families* CLL families References

All ages 33/1242 (2Æ7%) 8/65 (12Æ3%) 7/167 (4Æ1%) Caporaso, N., Goldin, L., Plass, C., Calin, G., Marti, G., Bauer, S., <40 1/365 (0Æ3%) 2/17 (11Æ8%) 0/54 (0) Raveche, E., McMaster, M.L., Ng, D., Landgren, O. & Slager, S. 40–60 10/457 (2Æ1%) 3/30 (10Æ0%) 2/81 (2Æ5%) (2007) Chronic lymphocytic leukemia genetics overview. British >60 22/420 (5Æ2%) 3/18 (16Æ7%) 5/32 (15Æ6%) Journal of Haematology, 139, 630–634. Chen, Z., Le Paslier, D., Dausset, J., Degos, L., Flandrin, G., Cohen, D. *de Tute et al (2006). & Sigaux, F. (1987) Human gamma genes are frequently Present study. rearranged in B-lineage acute lymphoblastic leukemias but not in chronic proliferations. The Journal of Experimental Medicine, 165, 1000–1015. families is probably related to the high prevalence of the MBL Dagklis, A., Fazi, C., Sala, C., Cantarelli, V., Scielzo, C., Massacane, R., in these individuals (Marti et al, 2007), our results suggest that Toniolo, D., Caligaris-Cappio, F., Stamatopoulos, K. & Ghia, P. in older first-degree relatives of patients with sporadic CLL, the (2009) The immunoglobulin gene repertoire of low-count CLL-like risk of MBL detection is as high as in older first-degree relatives MBL is different from CLL: diagnostic implications for clinical from CLL families. Therefore, the risk of developing clinical monitoring. Blood, 114, 26–32. CLL could be similar between these two categories. Dyer, M.J. (1989) T-cell receptor delta/alpha rearrangements in lym- Lastly, the prevalence rate in older first-degree relatives of phoid neoplasms. Blood, 74, 1073–1083. Fung, S.S., Hillier, K.L., Leger, C.S., Sandhu, I., Vickars, L.M., Gal- sporadic CLL indicated the fact that we could have a larger braith, P.F., Li, C.H. & Leitch, H.A. (2007) Clinical progression and population of individuals to search for MBL, provided that outcome of patients with monoclonal B-cell lymphocytosis. Leuke- MBL in general population is an uncommon finding and that mia & Lymphoma, 48, 1087–1091. the high prevalence of MBL in familial CLL is counterbalanced Ghia, P., Prato, G., Scielzo, C., Stella, S., Geuna, M., Guida, G. & by the low number of CLL families around the world (Yuille Caligaris-Cappio, F. (2004) Monoclonal CD5+ and CD5- B-lym- et al, 2000). In the present study, only one familial CLL was phocyte expansions are frequent in the peripheral blood of the detected in 43 investigated. Consecutively, a more common elderly. Blood, 103, 2237–2342. source of CLL-like cells to examine susceptibility genes in CLL Kerlan-Candon, S., Soua, Z., Lefranc, M.P., Clot, J. & Eliaou, J.F. and, moreover, the aetiologic factors of MBL, is readily (1998) Detection of receptor gene rearrangements in lym- available. phoproliferative malignancies by fluorescent polymerase chain In summary, MBL is a ‘unique’ condition with the same reaction. Tissue Antigens, 51, 20–29. Landgren, O., Albitar, M., Ma, W., Abbasi, F., Hayes, R.B., Ghia, P., biological characteristics, but with distinct prevalence rates in Marti, G.E. & Caporaso, N.E. (2009) B-cell clones as early markers healthy individuals from families with sporadic CLL cases and for chronic lymphocytic leukemia. New England Journal of Medicine, from those with familial CLL, and also in different age groups. 12, 659–667. Moreover, MBL is immunophenotypically and genetically Linet, M.S., Schubauer-Berigan, M.K., Weisenburger, D.D., Richard- similar to CLL and even ‘abnormal’ clonal rearrangements of son, D.B., Landgren, O., Blair, A., Silver, S., Field, R.W., Caldwell, G., the T-cell receptor genes can also be present in MBL as Hatch, M. & Dores, G.M. (2007) Chronic lymphocytic leukemia: an previously detected in some cases of CLL. At last, in subjects overview of aetiology in light of recent developments in classification aged more than 60 years, our results indicate that MBL is as and pathogenesis. British Journal of Haematology, 139, 672–686. frequent in first-degree relatives of patients with familial CLL Marti, G.E., Carter, P., Abbasi, F., Washington, G.C., Jain, N., Zenger, as in first-degree relatives of patients with sporadic CLL. V.E., Ishibe, N., Goldin, L., Fontaine, L., Weissman, N., Sgambati, M., Fauget, G., Bertin, P., Vogt, R.F., Slade, B., Noguchi, P.D., Steler-Stevenson, M.A. & Caporaso, N. (2003) B-cell monoclonal Acknowledgements lymphocytosis and B-cell abnormalities in the setting of familial B-cell chronic lymphocytic leukemia. Cytometry. Part B, Clinical The authors thank to Aglair B. Garcia, Denise A. P. Gallo, Cytometry, 52B, 1–12. Marli H. Tavela, Ame´lia G. Arau´ jo and Rosane de Paula Marti, G.E., Rawstron, A.C., Ghia, P., Hillmen, P., Houlston, R.S., Kay, Queiro´s for the technical assistance. N., Schleinitz, T.A. & Caporaso, N. (2005) Diagnostic criteria for monoclonal B-cell lymphocytosis. British Journal of Haematology, 130, 325–332. Authorship Marti, G., Abbasi, F., Raveche, E., Rawstron, A.C., Ghia, P., Aurran, T., D.M.M, designed research, recruited subjects, performed flow Caporaso, N., Shim, Y.K. & Vogt, R.F. (2007) Overview of mono- cytometry analysis and wrote the paper. S.J.I, was responsible clonal B-cell lymphocytosis. British Journal of Haematology, 139, for the recruitment of subjects. C.A.S, performed PCR analysis 701–708. Matutes, E., Owusu-Ankomah, K., Morila, R., Garcia, M.J., Houlihan, and wrote the paper. F.M.O, performed FISH analysis and A., Que, T.H. & Catovsky, D. (1994) The immunological profile of wrote the paper. E.M.R, analysed and interpreted data and

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