Bone Marrow Histology in Monoclonal B-Cell

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390 Am J Clin Pathol 2013;139:390-395390 DOI: 10.1309/AJCPPHSUQM8XBJH7

© American Society for Clinical Pathology

Hematopathology / Bone Marrow Histology in MBL

/

Bone Marrow Histology in Monoclonal B-CellLymphocytosis Shows Various B-Cell Infiltration Patterns

Ulla Randen, MD,1,2 Anne M. Tierens, MD, PhD,1 Geir E. Tjønnfjord, MD, PhD,2,3

and Jan Delabie, MD, PhD1,2

Key Words: Monoclonal B-cell lymphocytosis; MBL; CLL; Bone marrow histology

DOI: 10.1309/AJCPPHSUQM8XBJH7

A b s t r a c t

Monoclonal B-cell lymphocytosis (MBL) is defined

as less than 5 × 109 /L monoclonal B cells in the blood of

otherwise healthy patients and is detected in 5% to 10%

of individuals older than 60 years. The bone marrow

findings in MBL have hitherto not been systematically

described. We have studied the histologic and

immunophenotypic findings in paired trephine biopsy

specimens and bone marrow aspirates of 26 patientswith MBL. Abnormal lymphoid infiltration was detected

in trephine biopsy specimens of 20 patients and was

demonstrated by flow cytometry of bone marrow

in all 26 patients. Three infiltration patterns were

discerned: focal interstitial lymphoid infiltration, focal

rounded and nonparatrabecular lymphoid aggregates,

and discrete diffuse lymphocytosis. Focal interstitial

lymphoid infiltration was seen only in patients with

chronic lymphocytic leukemia (CLL)–like or atypical

CLL-like MBL, whereas the other patterns were not

related to a particular MBL immunophenotype. Our results show that most patients with MBL also have

bone marrow lymphoid infiltration. The latter should

be distinguished from lymphoma infiltration by clinical

correlation.

Monoclonal B-cell lymphocytosis (MBL) was first rec-

ognized as an indolent variant of chronic lymphocytic leuke-

mia (CLL) in patients with Rai stage 0 disease. The patients

showed no disease progression, even after a period of more

than 24 years.1 A similar indolent form of CLL was later

described in otherwise healthy family members of patients

with CLL and in cohort studies of outpatients.2-5 The condi-

tion has since been called MBL, and its natural course and

genetics have been studied extensively.6 MBL is now definedas less than 5 × 109/L monoclonal B cells in the blood of

otherwise healthy patients. Large population-based screening

studies have revealed that the prevalence of MBL increases

with age, with a frequency of up to 14% at an age of more

than 62 years.7-9 Virtually all patients with clinically evident

CLL had a preceding MBL, whereas the risk for patients with

CLL-like MBL to develop leukemia is about 1% per year.7,8

MBL likely arises through chronic stimulation of a limited

B-cell repertoire in older individuals. Thus, it is a phenom-

enon associated with senescence of the lymphoid system.10

MBL shows genetic changes such as 13q14 deletion, trisomy12, and deletion of 17p and 11q, as also seen in CLL.7 CD38

and ZAP-70 expression, VH gene usage, and cytogenetic

abnormalities have not allowed prediction of the transition of

MBL to CLL, but B-cell counts, especially low-count MBL,

have been associated with a very low risk of progression to

CLL.11,12 In addition to the more frequent CLL-like immuno-

phenotype of MBL, characterized by CD5 and CD23 expres-

sion and weak expression of CD20 and immunoglobulins, an

atypical CLL-like phenotype with moderate or strong CD20

and immunoglobulin expression is also recognized. Further-

more, MBL with a non–CLL-like phenotype, characterized

Upon completion of this activity you will be able to:• recognizethebonemarrowtrephinehistologicfindingsinmonoclonal

B-cell lymphocytosis.•advisetheclinicianabouttheimplicationofdiagnosinglesionsin

bone marrow trephines that are consistent with monoclonal B-celllymphocytosis.

TheASCPisaccreditedbytheAccreditationCouncilforContinuingMedicalEducationtoprovidecontinuingmedicaleducationforphysicians.TheASCPdesignatesthisjournal-basedCMEactivityforamaximumof1AMA PRA Category 1 Credit ™perarticle.Physiciansshouldclaimonlythecreditcommensuratewiththeextentoftheirparticipationintheactivity.ThisactivityqualifiesasanAmericanBoardofPathologyMaintenanceofCertificationPartIISelf-AssessmentModule.

Theauthorsofthisarticleandtheplanningcommitteemembersandstaff

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Am J Clin Pathol 2013;139:390-395 391391 DOI: 10.1309/AJCPPHSUQM8XBJH7 391


Hematopathology / Original Article

by the absence of CD5 expression, has also been described.5,6

In view of the frequency of MBL in the general population,

MBL is often seen in combination with other diseases, includ-

ing those for which a trephine biopsy regularly is performed

as part of the diagnostic workup. Trephine biopsy is part of

the panel of tests performed for a large number of diseases,

including primary nonmalignant as well as malignant hema-

tologic diseases, nonhematologic malignancies, and certain

infectious diseases. Hitherto, there have been no systematic

studies of bone marrow infiltration, including bone marrow

histologic findings, in patients with MBL despite its high fre-

quency in the population. We therefore investigated whether

MBL involved the bone marrow and, if so, what its histology

was like and whether it could be differentiated from bone mar-

row involvement by lymphoma. We studied the bone marrow

histology in 26 patients with well-documented MBL.

Materials and Methods

Patient Selection

Patients were selected from the database of the Depart-

ment of Pathology, Oslo University Hospital, Oslo, Norway.

Only patients with an established MBL diagnosed between

2008 and 2011 and from whom a trephine biopsy was

procured at the time of MBL diagnosis were selected for

this study. All patients with non-Hodgkin lymphoma were

excluded from the study, with the exception of 2 cases in

which lymphoma involvement was easy to distinguish fromMBL by morphology and immunohistochemistry. In total,

26 patients were retrieved from the database, including 18

men and 8 women with a mean age 70 years (range, 50-94

years). The patients underwent bone marrow examination as

part of the clinical workup of their initial symptoms or pri-

mary disease. In 17 patients who presented with nonspecific

symptoms such as fatigue, weight loss, or fever, no disease

was eventually diagnosed. Chronic myeloproliferative disease

was diagnosed in 4 patients and myelodysplastic syndrome

in 1 patient. Three patients had a plasma cell neoplasia, 1

had a primary cutaneous follicular lymphoma, and 1 had ahepatosplenic T-cell lymphoma. One patient had renal cell

carcinoma. A bone marrow aspirate had also been procured

in all 26 patients.

Flow Cytometry

Flow cytometry analyses were performed on blood and

bone marrow samples from all 26 patients. Samples were

anticoagulated with heparin. Between 0.5 and 1 × 106 cells

were stained for surface antigens. For samples analyzed before

2011, a 4-color analysis13 was performed with the following

antibody combinations labeled with fluorescein isothiocyanate

(FITC)/phycoerythrin (Pe)/peridinin-chlorophyll cyanine 5.5

(PercPCY5.5)/allophycocyanin (APC): (1) CD20/CD5/CD19/

CD43, (2) FMC7/CD23/CD19/CD5, (3) Igk/Igl/CD20/CD19,

(4) CD22/CD24/CD19/CD34, and (5) cyBcl2/CD10/CD19/

CD38. From 2011, an 8-color flow cytometry analysis14 was

used with the following antibody combinations labeled with

Pacific Blue/e450 (PB/e450), Krome Orange (KO), FITC/

Pe/PercPCy5.5/phycoerythrin cyanine 7(PeCy7)/APC/APC

Hilite7, or APC/cyanine 7 (APCH7/cy7): (1) CD20+CD4/

CD45/CD8+Igl/CD56+Igk/CD5/CD19+TCRγδ/CD38 and

(2) CD20/CD45/CD23/CD10/CD79b/CD19/CD200/CD43.

All antibodies for the 4-color panels were purchased from

Becton-Dickinson (San Jose, CA) except anti-FMC7, CD22,

CD23, and anti-Bcl2, which were purchased from DAKO

(Glostrup, Denmark). For the 8-color panels, CD56, CD5,

CD3, and 79b were purchased from Becton-Dickinson; CD23

from DAKO; CD200 from eBioscience (San Diego, CA);

CD8, k, and l from Cytognos (Salamanca, Spain); and the rest

from Beckman Coulter (Brea, CA). Flow cytometry analysis

was performed on a FACSCalibur or LSRII instrument (Bec-

ton-Dickinson) using CellQuest Pro and FACSDiva software

(Becton-Dickinson), respectively.

Immunohistochemistry

The primary antibodies used for immunophenotypic

analysis of the lymphoid infiltrates were anti-CD45, anti-

CD20, anti-Bcl6, anti-Mum1, anti-IgA, anti-IgD, anti-IgG,

anti-IgM, and anti-Ki67 (all from DAKO); anti-CD10, anti-

Bcl2, anti-CD21, anti-CD23, anti-CD5, anti-CD43, and anti–

cyclin D1 (all from Novocastra Laboratories, Newcastle uponTyne, UK); anti-PAX5 (Becton-Dickinson); and anti-CD3

(Thermo Fisher Scientific, Fremont, CA). For all antibodies,

heat-induced epitope retrieval was performed in a microwave

oven by heating the slides for 5 minutes at 750 W and sub-

sequently for 15 minutes at 500 W in retrieval buffer. The

detection system EnVision (DAKO) was used for all antibod-

ies. The stain was developed with 3,3'-diaminobenzidine and

H2O

2, and the slides were counterstained with hematoxylin.

The immunostaining was performed in a DAKO Autostainer

(DAKO) according to the instructions of the manufacturer.

Results

Flow cytometry analysis revealed a CLL-, an atypical

CLL-, or a non–CLL-like immunophenotype MBL in 11, 5,

and 10 patients, respectively ❚Table 1❚. Monoclonal B cells

were also detected in the bone marrow samples of all 26

patients. The immunophenotypes of the cells in the bone mar-

row were identical to those of the respective blood samples.

The trephine biopsy specimens showed no evidence of

abnormal lymphoid infiltration in 6 of 26 patients. Infiltration





Randen et al / Bone Marrow Histology in MBL

of the bone marrow by small B cells was found in 20 patients.

Three infiltration patterns were discerned: 7 patients showed

small interstitial foci, varying from 1 to 5, with monotonous

small lymphoid cells with a rounded nucleus. At the periphery

of these foci, cells infiltrated between fat cells in small rows

❚Image 1A-D❚. Eight patients showed more rounded lym-

phoid foci, varying from 1 to 9, without interstitial infiltration

between fat cells at the periphery. These lymphoid foci con-

sisted of small lymphoid cells with a slightly more irregular

nuclear contour, as well as a few scattered immunoblasts and

histiocytic cells ❚Image 1E-H❚. Centrally located tiny germinalcenters could occasionally be observed (Image 1H). The latter

were highlighted by CD21 staining. Finally, 5 patients showed

a discrete diffuse lymphocytosis that was only discerned upon

immunohistochemical staining for CD20. Diffuse B-cell lym-

phocytosis in patients with MBL could be distinguished from

the presence of hematogones by the homogeneous small size

of B cells in the former and by the variable small to medium-

large size in the latter. In addition, hematogones showed a

variable CD20 expression in contrast to the homogeneous

CD20 expression seen with MBL. Intrasinusoidal infiltration

was not seen in any of the cases.

Of interest, there was a correlation between the infiltra-

tion pattern and the immunophenotype of MBL (Table 1). The

interstitial pattern was exclusively seen in patients with MBL

with a CLL-like or an atypical CLL-like immunophenotype.

The 2 other patterns were most commonly seen in non–CLL-

like MBL, although these patterns were also observed in 3

patients with a CLL-like or an atypical CLL-like MBL.

Discussion

With the extensive use of highly sensitive multiparameter

flow cytometry analyses of blood and bone marrow samples,

MBL is increasingly being detected.9,15 The etiology of MBL

is not clear, but it is hypothesized that it results from chronic

and persistent antigen stimulation.10,16 CLL-like and atypical

CLL-like MBL shows genetic changes such as chromosome

13q deletion, trisomy 12, and even chromosome 17p deletions

as well as stereotyped immunoglobulin receptors as seen in

CLL, adding to the argument that MBL is a precursor state of

CLL.7,12 Less is known about non–CLL-like MBL, although

genetic changes are also seen, albeit different from those

❚Table 1❚Infiltration Pattern and Immunophenotype of Bone Marrow B-Cell Infiltration in Patients With MBL

Infiltration No. of DiscretePatient No. Sex/Age, y Pattern Infiltrations Concurrent Disease

CLL-phenotype casesa

1 F/73 0 0 CMPD2 M/74 1 1 Primary cutaneous FL

4 M/67 0 0 ND12 M/80 1 2 MDS13 F/58 2 1 ND14 F/50 1/3 4 ND20 F/72 1 3 ND24 M/76 0 0 ND25 M/89 2 1 Plasma cell neoplasia22 M/69 0 0 ND23 M/82 1 2 ND

Atypical CLL-phenotype casesb 6 M/64 0 0 ND7 M/70 1 3 ND8 M/56 3 0 CMPD15 M/60 2 7 ND26 M/55 1 5 ND

Non–CLL-phenotype casesc 3 F/74 0 0 CMPD5 F/63 3 0 ND9 M/82 3 0 Renal cell carcinoma10 M/88 2 1 CMPD11 F/79 3 0 Plasma cell neoplasia16 F/75 2 2 ND17 M/67 2 1 Plasma cell neoplasia18 M/94 2 9 ND19 F/81 2 1 Hepatosplenic T-cell lymphoma21 M/69 3 0 ND

Infiltration patterns: 0, no infiltration; 1, interstitial infiltration; 2, circumscribed intraparenchymatous lymphoid aggregates; 3, diffuse infiltration. CLL, chronic lymphocyticleukemia; CMPD, chronic myeloproliferative disease; FL, follicular lymphoma; MBL, monoclonal B-cell lymphocytosis; MDS, myelodysplastic syndrome; ND, no disease.

a CD20dim+, CD5+, CD23+, Ig light chain dim+. b CD20+, CD5+, CD23+, Ig light chain+.c CD20+, CD5–, CD23–, Ig light chain+.






demonstrated in CLL-like MBL.17 Whether non–CLL-like

MBL is a precursor state for non-Hodgkin lymphoma—in

particular, marginal zone lymphoma or lymphoplasmacytic

lymphoma and perhaps also diffuse large B-cell lymphoma,

as the immunophenotype and genetic changes seem to indi-

cate—remains to be demonstrated.18 Whether and how MBL

affects the bone marrow was hitherto poorly described except

for 1 study published as an abstract.19 However, knowledge

about bone marrow infiltration in MBL is of potential inter-

est to avoid diagnosis of marrow infiltration with lymphoma.

We therefore studied trephine biopsy specimens by histology

and immunohistochemistry in 26 individuals with established

MBL. We reviewed the flow cytometry findings in peripheral

blood and bone marrow in all patients.

Histology and immunohistochemistry highlighted abnor-

mal lymphoid infiltration in the bone marrow of 20 patients,

displaying patterns that were indistinguishable from limited

infiltration by CLL or marginal zone lymphoma. CLL or

small lymphocytic lymphoma shows nodular or diffuse

non-paratrabecular interstitial infiltration with monotonous

small lymphoid cells variably admixed with paraimmuno-

blasts.20 Occasionally, more limited focal patchy interstitial

A B

C D

❚Image 1❚ A-D, Bone marrow lymphoid infiltration in chronic lymphocytic leukemia (CLL)–type monoclonal B-cell lymphocytosis

(MBL). Focal interstitial infiltration by small lymphoid cells with rounded nuclei is shown (A, H&E, ×400, patient 12; B, H&E,

×400, patient 14). Immunohistochemical staining for CD20 highlights the focal interstitial infiltration pattern (C, ×200, patient 23)

and cell-poor diffuse infiltration pattern that can be seen in MBL (D, ×200, patient 13).





Randen et al / Bone Marrow Histology in MBL

infiltrates are seen in CLL, indistinguishable from the pat-

tern of infiltration reported here for MBL with a CLL-like

or an atypical CLL-like immunophenotype.20 Marginal

zone lymphoma in the bone marrow most frequently shows

focal, more rounded nonparatrabecular infiltrates with small

lymphoid cells often admixed with some immunoblasts and

histiocytic cells as well as follicular dendritic cells.20,21

This is also one of the patterns seen in MBL with a non–

CLL-like immunophenotype, although this pattern could

occasionally be observed in MBL with a CLL-like and an

atypical CLL-like immunophenotype. Of interest, diffuse

but limited B-cell infiltration was almost exclusively seen

in MBL with a non–CLL-like immunophenotype. This

infiltration pattern is distinct from the diffuse but intrasinu-

soidal infiltration pattern often seen in splenic marginal

zone lymphoma.20 Intrasinusoidal infiltration was not seen

in any of the MBL cases.

There was perfect agreement between the immunophe-

notype of MBL in peripheral blood and the monoclonal B

cells in the bone marrow. In addition, immunohistochemical

findings were also in agreement with the immunopheno-

types detected by flow cytometry. This supports the finding

❚Image 1❚ (cont) E and F, Lymphoid infiltration seen in non–CLL-type MBL (E, H&E, ×400, patient 16; F, anti-CD20, ×400,

patient 15); there is focal but more clustered infiltration in contrast to the typical CLL-type MBL. There are small lymphoid cells

with nuclei with slightly irregular contours admixed with the same few immunoblasts (E). G, Lymphoid infiltration in a case of

atypical CLL-type MBL (H&E, ×400, patient 16). In this case, small lymphoid cells are admixed with epithelioid histiocytic cells.

H, Focal cluster of B cells in a patient with atypical CLL-type MBL (anti-CD20, ×200, patient 7) displaying a small network of

follicular dendritic cells in the background of the lymphoid focus as highlighted by CD21 expression (not illustrated).

E F

G H






that lymphoid infiltration seen in bone marrow is the tissue

counterpart of MBL.

The frequent involvement of bone marrow in patients

with MBL displaying an infiltration pattern and immunophe-

notype that is indistinguishable from that of limited infiltration

with lymphoma, especially CLL or marginal zone lymphoma,

is of diagnostic importance. Our results indicate that a final

diagnosis of lymphoma in the bone marrow should not be

made in the absence of an established lymphoma diagnosis or

in the absence of adequate clinical information. Whether the

patient with limited lymphoid infiltration in the bone marrow,

originally taken as part of the investigation for other diseases,

should be screened for lymphoma and how extensive this

screening should be is a matter of discussion. Noninvasive

investigations with abdominal ultrasound and chest x-ray

in addition to a complete blood cell count, whenever these

have not been performed as part of the investigations for the

patient’s primary disease, are most likely indicated, as recom-

mended for MBL.10,22

Whether the extent of bone marrow infiltration with

MBL correlates with increased progression toward overt

clinical disease is of interest but still needs to be studied. This

question may be answered by studying a larger cohort of

patients with a longer follow-up time.

From the 1 Department of Pathology and the 3 Department of

Hematology, Oslo University Hospital, Oslo, Norway; and 2 Institute of Clinical Medicine, University of Oslo, Oslo, Norway.

This study was supported by a grant from the Norwegian

Cancer Society.

Address reprint requests to Dr Randen: Dept. of Pathology,

Oslo University Hospital, Ullernchausséen 70, 0310 Oslo,

Norway; e-mail: [email protected].

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Bone Marrow Histology in Monoclonal B-Cell