Published Ahead of Print on May 22, 2015, as doi:10.3324/haematol.2015.126656. Copyright 2015 Ferrata Storti Foundation.
Primary diffuse large B cell lymphoma associated with Clonally-related monoclonal B lymphocytosis indicates a common precursor cell by Agnieszka Malecka, Anne Tierens, Ingunn Østlie, Roland Schmitz, Gunhild Trøen, Signe Spetalen, Louis M. Staudt, Erlend Smeland, Harald Holte, and Jan Delabie Haematologica 2015 [Epub ahead of print] Citation: Malecka A, Tierens A, Østlie I, Schmitz R, Trøen G, Spetalen S, Staudt LM, Smeland E, Holte H, and Delabie J. Primary diffuse large B cell lymphoma associated with Clonally-related monoclonal B lymphocytosis indicates a common precursor cell. Haematologica. 2015; 100:xxx doi:10.3324/haematol.2015.126656 Publisher's Disclaimer. E-publishing ahead of print is increasingly important for the rapid dissemination of science. Haematologica is, therefore, E-publishing PDF files of an early version of manuscripts that have completed a regular peer review and have been accepted for publication. E-publishing of this PDF file has been approved by the authors. After having E-published Ahead of Print, manuscripts will then undergo technical and English editing, typesetting, proof correction and be presented for the authors' final approval; the final version of the manuscript will then appear in print on a regular issue of the journal. All legal disclaimers that apply to the journal also pertain to this production process.
1
Primary Diffuse Large B cell lymphoma Associated with Clonallyrelated Monoclonal B lymphocytosis indicates a common precursor cell. 1
2
1
3
1
Agnieszka Malecka , Anne Tierens , Ingunn Østlie , Roland Schmitz , Gunhild Trøen ,
1
3
4,5,
Signe Spetalen , Louis M. Staudt , Erlend Smeland
1Department of 2Laboratory
5,6 and
Harald Holte
2,5
Jan Delabie
Pathology, Oslo University Hospital, 0310 Oslo, Norway
Medicine Program, University Health Network and Laboratory Medicine
and Pathobiology, University of Toronto, Toronto, Ontario M5G 2C4, Canada
3Lymphoid
Malignancies Branch, Center for Cancer Research, National Cancer Institute,
National Institutes of Health, Bethesda, Maryland 20892, U.S.A
4Institute 5Center
for Cancer Research, Oslo University Hospital, 0310 Oslo, Norway
for Cancer Biomedicine, University of Oslo, 0310 Oslo, Norway
6Department of
Running head:
Oncology, Cancer Clinic, Oslo University Hospital, 0310, Oslo, Norway
Clonally related DLBCL and MBL
Corresponding Author:
Jan Delabie, M.D., Ph.D.
Laboratory Medicine and Pathobiology
University Health Network and University of Toronto
200 Elizabeth Street
Toronto, ON, M5G 2C4
Canada
E-mail:
[email protected]
Tel: 416 3405239
Fax: 416 3405543
2
Word Counts:
Text: 1325
Number of Figures: 1
Number of Tables: 1
Number of References: 19
Acknowledgements:
This study was supported by Health Region Authority South-East Norway and by the
Norwegian Cancer Society.
3
Bone
marrow
monoclonal
small
B
cell
infiltration
(MSBC)
associated
with
monoclonal B lymphocytosis (MBL), is present at a higher frequency in patients with
diffuse large B cell lymphoma (DLBCL). We have prospectively collected blood and bone
marrow
samples
in
patients
with
primary
DLBCL
at
diagnosis
to
study
the
clonal
relationship of MBL/MSBC with the paired DLBCL. MBL/MSBC were detected in 6/19
patients,
of
whom
germinal center
5
with
DLBCL
B cell (GCB)
with
origin.
activated
B
cell
origin
(ABC)
and
MBL/MSBC were clonally related
to
one
the
with
paired
DLBCL in 3/6 patients as demonstrated by rearranged immunoglobulin heavy chain
gene
sequence
lymphocytic
analysis.
leukemia
MBL/MSBC
(CLL)-like,
a
in
these
three
non-CLL-like
patients
and
a
showed
germinal
a
chronic
center
cell-like
immunophenotype, respectively. The former two MBL/MSBC immuno-phenotypes were
associated
rearranged
with
DLBCL-ABC,
immunoglobulin
the
latter
genes
with
were
DLBCL-GCB.
demonstrated
Similar
for
the
but
three
not
identical
patients
with
MBL/MSBC and paired DLBCL that were not clonally related. These results suggest that
a subset of DLBCL may arise from MBL/MSBC.
The
most
common
type
of
DLBCL,
DLBCL
not
otherwise
specified
(NOS)
is
subdivided according to cell of origin, either from activated B cells or from germinal
center
1,2
B cells.
characterized
DLBCL-ABC is genetically different compared to DLBCL-GCB and
by
chronic
active
B
cell
receptor
and
Toll-like
receptor
is
signaling
pathways, that may be targeted with novel drugs.
We previously reported a high incidence of MSBC in bone marrow and MBL in the
blood of DLBCL patients, but due to the lack of materials the clonal relationship between
3 DLBCL-ABC
the two lymphoproliferative diseases could not be studied but for one case.
showed
a
higher
3
respectively.
frequency
Also,
of
MBL/MSBC
MBL/MSBC
was
more
than
DLBCL-GCB,
frequently
of
28,2%
non-chronic
versus
3,7%,
lymphocytic
leukemia (CLL) type than of CLL type, the latter being the most frequent MBL type in the
4
general population.
more than 10%.
with
CLL-type
5
MBL has been detected in elderly patients, with an incidence rate of
6,7
CLL-type MBL is a precursor lesion for CLL.
MBL
do
not
develop
CLL.
Non-CLL-type
MBL
However, most patients
has
hitherto
not
been
demonstrated to be a precursor lesion for lymphoma or leukemia, but was proposed in
8
one study to represent a low-grade lymphoma of the bone marrow.
4
Bone marrow (10 ml) and blood samples (20 ml) of patients with primary DLBCL
at diagnosis were prospectively collected to detect MBL/MSBC and study whether the
latter cells were clonally related to the paired DLBCL. Samples were ultimately collected
for 19 patients. All patients were treated at the Norwegian Radium Hospital, Norway
(Supplemental table 1). Diagnostic lymphoma tissue and bone marrow trephine biopsy
was available for all patients. The study was approved by the Regional Committee for
Medical
and
Health
Professional
Research
Ethics
of
South-East
Norway
(reference
number 2010/3241).
All
lymphoma
appropriate
and
bone
marrow
immunohistochemical
trephine
analysis
was
biopsies
performed
in
were
all
reviewed
cases.
and
Lymphomas
were diagnosed according to the W.H.O. classification and the DLBCL cell of origin was
1,9
studied using the Hans algorithm.
Eight-color
flow
cytometry
analysis
was
used
with
the
following
antibody
combinations labeled with Pacific Blue/ e450 (PB/e450), Krome Orange (KO), FITC/ Pe
/
PercPCy5.5/
Phycoerithrin
cyanine
7(PeCy7)/APC/
APC
Hilite7
or
APC/cyanine7
λ/CD56+/Igκ/CD5/CD19+TCRγδ/CD38; (2)
(APCH7/cy7): (1) CD20+CD4/CD45/CD8+Ig
CD20/CD45/CD23/CD10/CD79b/CD19/CD200/CD43.
Flow
cytometry
analysis
was
performed on a LSRII instrument (Becton-Dickinson), using FACSDiva software (Becton-
Dickinson).
If
monoclonal
B
cells
were
sorted with high pressure settings
present
stained
using a FACS
samples
were
subsequently
Aria IIu High speed sorter (Becton
Dickinson) equipped with 408 nm, 488 nm and 633 nm lasers. Selection of MBL/MSBC
for
sorting
was performed using Becton Dickinson FACSDiva software, starting with
gating of viable cells using the forward scatter versus side scatter dot plot. Subsequently,
T-cells and B-cells were gated out using a CD5 versus CD19 dot plot. Finally, MBL/MSBC
were
separated
from
polyclonal
B-cells
taking
advantage
of
the
aberrant
B-cell
phenotypes identified by flow cytometry analysis. Cells were collected in PBS or RLT
plus
lysis
buffer
(Qiagen,
Germany).
Six
of
the
19
patients
with
DLBCL
showed
MBL/MSBC. In accordance with our previous study, five of six patients had DLBCL-ABC
and one had DLBCL-GCB. MSBC in the bone marrow was associated with MBL in all
patients
(Supplemental
table
1).
The
close
association
between
MSBC
in
10,11
marrow and MBL in peripheral blood has previously been demonstrated.
the
bone
Also, the
bone marrow trephine biopsies of the six patients showed in total one to three foci with
small B lymphoid cells with infiltration patterns as previously described in patients with
5
10,11
None of the patients showed
cell
lymphoma.
MBL (Figure 1 and Supplemental Figures 1 and 2).
histologic
bone
marrow
infiltration
with
large
B
Also,
MBL/MSBC
identified by flow cytometry showed a forward scatter overlapping with that of small
polytypic B-lymphocytes in the same sample, indicating the small size of these cells
(Supplemental Figure 3). The bone marrow trephine biopsies of the 13 patients without
MBL/MSBC as detected by flow cytometry,
did not show small B cell
aggregates by
immunohistology.
In order to study the clonal relationship between MBL/MSBC and DLBCL paired
samples, rearranged IGH gene sequences were analysed. Rearranged IGH genes from
sorted
MBL/MSBC
were
amplified
from
DNA
using
the
IGH
Somatic
Hypermutation
Assay v2.0 (Invivoscribe Inc., San Diego, CA). For DLBCL samples, formalin-fixed tissue
was analysed using primers complimentary to IGHV framework 1, 2 and 3 as described
12
before.
For one patient, frozen DLBCL tissue was available and was studied as for
MBL/MSBC.
The
PCR
products
were
subsequently
sequenced
using
the
BigDye®
Terminator v1.1 Cycle Sequencing Kit (Life Technologies, Carlsbad, CA) and the primers
from
the
IGH
Somatic
Hypermutation
Assay
v2.0
kit
(Invivoscribe
Inc.).
The
International Immunogenetics Information System web-based software (www.imgt.org)
was used to analyze the rearranged IGH sequences. The entire analysis was repeated
twice.
In
addition,
sequencing
was
repeated
with
IGHV
family-specific
12
primers.
Rearranged IGHV sequences of MBL/MSBC and DLBCL revealed a clonal relationship in
three of six paired samples. The samples of patients one and two showed 94.27% and
94.59 %
paired
homology
MBL/MSBC
immunophenotype
to
germline
and DLBCL
(Table
1).
IGHV
genes, respectively.
samples.
The
Patient
paired
one
DLBCL
Mutations
were similar
showed MBL/MSBC
of
this
patient
with
showed
in
a CLL
an
ABC
immunophenotype without CLL immunophenotype. Patient two showed a non-CLL-type
MBL/MSBC
with
a
concordant
DLBCL-ABC
immunophenotype.
Patient
six
showed
shared as well as divergent somatic mutations in MBL/MSBC and DLBCL with the latter
showing more mutations (Table 1). The MBL/MSBC showed a GCB immunophenotype
as
did
the
paired
DLBCL.
The
GCB
immunophenotype
is
consistent
with
the
high
mutation rate and the presence of divergent mutations, suggesting on-going mutation.
Histologic review of the DLBCL sample did not reveal concurrent follicular lymphoma.
Also, only one focal paratrabecular small B-cell aggregate was seen in the bone marrow.
These
findings
were
not
diagnostic
of
concurrent
follicular
lymphoma.
Parallel
6
development
of
DLBCL
from
a
common
13 Case
demonstrated for follicular lymphoma.
progenitor
cell
has
previously
been
6 in our series may represent a variant of
the same process.
Taken
together,
a
common
clonal
origin
of
MBL/MSBC
and
DLBCL
was
demonstrated in 3/6 cases and strongly suggests that a subset of DLBCL may arise from
small precursor cells, with either a CLL, non-CLL or GC immunophenotype. The former
two are associated with DLBCL-ABC. One case of CLL-like MBL/MSBC, clonally related to
3
DLBCL-ABC was also demonstrated in our earlier publication.
IGHV gene usage was either the same or belonged to the same VH gene family for
paired MBL/MSBC and DLBCL of the three cases without clonal relationship. Whether
this
indicates
that
MBL/MSBC
and
DLBCL
arise
through
antigen-stimulation
with
subsequent selection of VH genes in these cases is an interesting hypothesis but needs as
yet to be demonstrated. Preferential VH gene use has previously been demonstrated in
14,15 More
DLBCL and may support this hypothesis.
samples are currently collected to
study the molecular genetics of paired MBL/MSBC and DLBCL.
In
conclusion,
primary
DLBCL
in
a
subset
MBL/MSBC, indicating a common progenitor cell.
of
patients
is
clonally
related
to
7
AUTHORSHIP AND DISCLOSURES
A.T., H.H, L.M.S and J.D. designed the study.
A.M, R.S, A.T., I.Ø and G.T designed and
performed the FACS sorting and immunoglobulin gene analysis. All authors have
critically reviewed the results and contributed to the writing of the manuscript.
The authors have no conflicts of interest to disclose.
8
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Swerdlow SH, Campo E, Harris NL, et al. (Eds): WHO Classification of Tumours of
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2.
Alizadeh
AA,
Eisen
MB,
Davis
RE,
et
al.
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types
of
diffuse
large
B-cell
lymphoma identified by gene expression profiling. Nature. 2000;403(6769):503-
511.
3.
Tierens AM, Holte H, Warsame A, et al. Low levels of monoclonal small B cells in
the bone marrow of patients with diffuse large B-cell lymphoma of activated B-
cell type but not of germinal center B-cell type. Haematologica. 2010;95(8):1334-
1341.
4.
Rawstron AC, Green MJ, Kuzmicki A, et al. Monoclonal B lymphocytes with the
characteristics of "indolent" chronic lymphocytic leukemia are present in 3.5% of
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5.
Nieto WG, Almeida J, Romero A, et al. Primary Health Care Group of Salamanca
for
the
Study
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MBL.
Increased
leukemia-like
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clones
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(12%)
healthy
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chronic
a
highly
sensitive multicolor flow cytometry approach. Blood. 2009;114(1):33-37.
6.
Rawstron AC, Bennett FL, O'Connor SJ, et al. Monoclonal B-cell lymphocytosis and
chronic lymphocytic leukemia. N Engl J Med. 2008;359(6):575-583.
7.
Landgren O, Albitar M, Ma W, et al. B-cell clones as early markers for chronic
lymphocytic leukemia. N Engl J Med. 2009;360(7):659-667.
8.
Xochelli A, Kalpadakis C, Gardiner A, et al. Clonal B-cell lymphocytosis
exhibiting immunophenotypic features consistent with a marginal-zone origin: is
this a distinct entity? Blood. 2014;123(8):1199-1206.
9
9.
Hans
CP,
Weisenburger
DD,
Greiner
TC,
et
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of
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molecular
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PCR primers and protocols for detection of clonal immunoglobulin and T-cell
receptor
gene
BIOMED-2
recombinations
Concerted
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BMH4-CT98-3936.
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2317.
11. Randen U, Tierens AM, Tjønnfjord GE, Delabie J. Bone marrow histology in
monoclonal B-cell lymphocytosis shows various B-cell infiltration patterns. Am J
Clin Pathol. 2013;139(3):390-395.
12. Nelson BP, Abdul-Nabi A, Goolsby C, Winter J, Peterson L. Characterization of
tissue findings in bone marrow biopsy specimens with small monoclonal B-cell
populations. Am J Clin Pathol. 2014;141(5):687-696.
13. Pasqualucci L, Khiabanian H, Fangazio M, et al. Genetics of follicular lymphoma
transformation. Cell Rep. 2014;6(1):130-140
14. Hsu FJ, Levy R. Preferential use of the VH4 Ig gene family by diffuse large-cell
lymphoma. Blood. 1995;86(8):3072-3082.
15. Sebastián
E,
Alcoceba
M,
Balanzategui
A,
et
al.
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driven origin and IGHV4-34 as a particular subgroup of the non-GCB subtype. Am
J Pathol. 2012;181(5):1879-1888.
10
TABLES
Table 1.
Immunophenotype and Immunoglobulin sequence characteristics of
MBL/MSBC and DLBCL
SAMPLE
Immunophenotype
COO
Patient 1
CD19+, CD20dim,
CLL-
MBL/MSBC
CD79b-, CD5+,
like
IGHV
IGHJ
IGHD
V-REGION
CLONAL
HOMOLOGY
IDENTITY
Yes
3-53
6
2-2
94.30%
ABC
3-53
6
2-2
94.30%
4-34
4
6-19
94.60%
ABC
4-34
4
6-19
94.60%
4-59
3
3-22
91.60%
ABC
4-34
4
6-25
96.10%
3-7
4
3-16
90.00%
ABC
3-23
5
6-13
94.40%
2-5
4
6-19
95.50%
ABC
2-5
4
2-15
100.00%
3-11
3
5-12
83.10%
3-11
3
5-12
77.50%
CDR3 AMINOACID SEQUENCE1
CARGDCSSTTCNILAVW
CD23dim, IgL+ DLBCL
CD20+, CD10-,
CARGDCSSTTCNILAVW
BCL6+, MUM1+, CD5-, CD23-, EBERPatient 2
CD19+, CD20+,
Non-
MBL/MSBC
CD79b+, CD5-,
CLL
Yes
CARGPRDDMAVALDNW
CD10-, CD23-, IgK+ DLBCL
CD20+, CD10-,
CARGPRDDMAVALDNW
BCL6+, MUM1+, CD5-, EBERPatient 3
CD19+, CD20+,
Non-
MBL/MSBC
CD79b+, CD5-,
CLL
No
CARAGQYYYDSSGYYAYAFDIW
CD23-, IgL+ Patient -3-
CD20+, CD10-,
DLBCL
BCL6-, MUM1-, CD5-
CARGCSVYGLVYW
, EBERPatient 4
CD19+, CD20dim,
CLL-
MBL/MSBC
CD79b-, CD5+,
like
No
CARYGRAVSIDYW
CD23dim, IgK+ DLBCL
CD20+, CD10-,
CAKECSNSWDTW
BCL6-, MUM1-, CD5, EBERPatient 5
CD19+, CD20+,
Non-
MBL/MSBC
CD79b+, CD5dim,
CLL
No
CAHIPYFHTSGRIFDYW
CD23-, IgK+ DLBCL
CD20+, CD10-,
CARPKKSLL*WW*LLFNV#FDYW
MUM1+, CD5-, CD23dim, EBERPatient 6
CD19+, CD20+,
GCB-
MBL/MSBC
CD79b+, CD5-,
like
Yes
CARIYRHSLDIW
CD23-, CD10+, IgK+ DLBCL
CD20+, CD10+,
GCB
CARFYRHAFDIW
MUM1-, CD5-, EBERComplete nucleic acid sequences are available from GenBAnk, with reference numbers KP734253 to KP734264. Markers in column
1
2 in bold are the ones used for fluorescence-activated cell sorting. Abbreviations: IGHV, IGHJ, IGHD: immunoglobulin heavy chain variable, junction and diversity genes, respectively; COO: cell of origin; CDR3: complimentarity determining region 3; MBL/MSBC: monoclonal small B cell infiltrate/monoclonal B cell lymphocytosis; DLBCL: diffuse large B cell lymphoma; CLL: chronic lymphocytic leukemia; ABC: activated B cell type; GCB: germinal center cell type
11
LEGEND TO THE FIGURES
FIGURE 1
Bone marrow trephine sections of patients 1 and 2, respectively, show a small
interstitial infiltrate with small lymphoid cells (panels A, B, H&E-stained sections, 400x).
The lymphoid cells are highlighted by anti-CD20 immunohistochemistry (panels C, D,
respectively, 400x). A rectal mucosa biopsy of patient 1 (panel E, H&E-stained section,
400x) and a gastric mucosa biopsy of patient 2 (panel F, H&E-stained section, 400x)
shows diffuse infiltration with large B cell lymphoma. The scale bar indicates 50
micrometer.
SUPPLEMENTAL DATA Supplemental Methods 1. Histology Hematoxylin and eosin-stained sections of formalin-fixed lymphoma tissue and of zincformalin-fixed and formic acid-decalcified bone marrow trephine biopsies were reviewed. Paraffin blocks were cut at 4–6 µm, dried overnight at 60°C and dewaxed in xylene prior to immunohistochemical staining. The following antibodies were used: antibodies against CD20, MUM1, BCL6, Ki67 (all from Dako Cytomation, Glostrup, Denmark), CD5, CD21, CD23, CD10, BCL2 (all from Novocastra, Newcastle, U.K.), CD3, cyclin D1 (Lab Vision/NeoMarkers, Fremont, CA), CD138 (Serotec, Kidlington, U.K. ) and PAX-5 (Becton Dickinson, Franklin Lakes, NJ). Visualization was performed using the EnVision® detection system (Dako Cytomation) according to the manufacturer’s instructions. Appropriate positive and negative controls were used. 2. Flow cytometry of blood and bone marrow samples Anti-CD56, anti-CD5, anti-CD3 and anti-CD79b were purchased from Becton-Dickinson (San José, CA, USA); anti-CD23 from Dako; anti-CD200 from eBioscience (San Diego, CA); anti-CD8, anti-Ig and anti-Ig from Cytognos (Salamanca, Spain) and the remaining of the antibodies (anti-CD4, anti-CD19, anti-CD20, anti-CD38, anti-CD43, anti-CD45 and anti-TCR from Beckman Coulter (Brea, CA). 3. Fluorescent activated cell sorting (FACS) of MBL/MSBC from blood or bone marrow Mononuclear cell suspensions were made of all bone marrow and blood samples using Leucosep® tubes (Greiner Bio-One North America, Inc.) according to manufacturer’s recommendations. Cells were resuspended in PBS supplemented with 1% FCS and 10% DMSO and were subsequently frozen using an isopropanol chamber and stored in liquid nitrogen until FACS analysis. Although all blood and bone marrow samples contained monoclonal B-cells as determined by prior flow cytometry analysis, cells were FACS-sorted
from either bone marrow (patients 1, 2, 4 and 6) or from blood (patients 3 and 5) depending on whichever sample volume was largest to be able to sort as many cells as possible. For FACS analysis, the mononuclear cell suspensions were thawed and divided in aliquots of 0,5-1,0 x 10^6 cells/tube. The cells were washed with 2000 µl PBS with 0,5 % BSA (PAA laboratories GmbH, Austria) and stained for surface antigens with the following antibodies: anti-CD45 (clone J.33, Beckman Coulter), anti -CD20 (clone B9E9(HRC20), Beckman Coulter), anti-CD19 (clone J3-119, Beckman Coulter), anti-CD5 (clone L17F12, Becton-Dickinson (San Jose, CA)) and anti-CD10 (clone HI10a, Becton Dickinson) anti-λ and anti-κ (polyclonal antibodies, Cytognos (Salamanca, Spain)). Antibodies were conjugated to either fluoresceine thyocyanate (FITC), phycoerythrine (Pe), peridinin chlorophyll proteincy5.5 (PerCP-Cy5.5), phycoerythrine cyanine 7 (PeCy7), allophycocyanin (APC), Pacific Blue or Krome Orange. After staining, the cell suspensions were incubated for 15 minutes in the dark at room temperature and washed with 2000 µl PBS supplemented with 0,5 % BSA. Aliquots with stained cell suspensions from each patient, respectively, were pooled and filtered through a 70 µm filter to remove cell clumps. Stained samples were sorted with high-pressure settings using a FACS Aria Ilu High speed sorter (Becton Dickinson) equipped with 408nm, 488nm and 633nm lasers. Selection of MBL/MSBC for sorting was performed using Becton Dickinson FACSDiva software, starting with the gating of viable cells using the forward scatter versus side scatter dot plot. Subsequently, CD45 bright, low side scatter events (i.e. lymphocytes) were selected. Then, CD5 positive and CD19 negative events (i.e. T cells) were gated out using a CD5 versus CD19 dot plot leaving only B cells. Finally, MBL/MSBC were separated from the polyclonal B cells taking advantage of the aberrant B-cell immunophenotype identified by prior flow cytometry analysis. The marker combination used for the latter is indicated for each patient in Table 1 of the main manuscript. 4. DNA extraction and whole genome amplification DNA from sorted MBL/MSBC was extracted using Qiagen AllPrep DNA/RNA Micro kit (Qiagen, Hilden, Germany) according the instructions of the manufacturer. Genomic DNA
was subsequently amplified using Illustra Ready-To-Go GenomiPhi V3 DNA Amplification Kit (GE Healthcare Life Sciences, U.K.). DNA from either formalin-fixed paraffin-embedded tissue or fresh frozen tissue of DLBCL samples, the latter only available for one case, was extracted using appropriate kits from Qiagen according to manufacturer’s recommendations. The concentration of all extracted nucleic acid was measured using a NanoDrop 2000 spectrometer (Thermo Scientific, Waltham, MA).
Legend to the supplemental figures Supplemental figure 1 Representative H&E-stained sections of the diffuse large B cell lymphoma biopsies of patients 3, 4 and 5 (panels A,B and C, respectively; 400x). Large atypical lymphoid cells are seen in all panels, with areas of necrosis in panels A and B. The scale bar indicates 50 micrometer.
Supplemental figure 2 Representative H&E-stained and anti-CD20 stained sections of bone marrow trephine biopsies of patient 4 (panels A,B), patient 5 (panels A,B) and patient 6 (panels A,B), respectively (400x). The sections illustrate infiltrates of small B lymphocytes. The infiltrates are in the intertrabecular parenchyma in patients 4 and 5. By contrast, a paratrabecular infiltrate is seen in patient 6. Histiocytes can be seen in the stroma of the lymphoid infiltrates in patients 4 and 5 (panels A,C). Since immunohistochemical analysis involves deeper sectioning and since the lymphoid infiltrates are small, the size and content of histiocytes differs between the H&E sections and the immunohistochemicallystained sections. The scale bar indicates 50 micrometer.
Supplemental figure 3 Analysis of small monoclonal B cells/monoclonal B cell lymphocytosis by flow cytometry for patients 1 to 6. The cells with an abnormal immunophenotype are highlighted in blue, polytypic B cells are highlighted in red. Panels A show the forward and side scatter of the cells, panels B show the staining with CD45. Forward and side scatter illustrate that the small monoclonal B cells/monoclonal B cell lymphocytosis share the same scatter characteristics of polytypic B lymphocytes present in the samples, consistent with the small
size of the monoclonal B cells. The gating strategy for isolation of MBL/MSBC by FACS is given at the bottom of the figure. Note that the yield in percentage of MBL/MSBC from sorted samples derived from mononuclear cell suspensions is different, usually higher, than the percentage of the respective cells detected by flow cytometry on lysed samples, still containing granulocytes, given in Table 1.
Supplemental Tables
Supplemental Table 1. Patient characteristics
Patient
Age
LDH1
Number of extranodal sites
Stage
WHO performance status
IPI2
Biopsy site
Blood lymphocyte count (x 109/L)
MBL3 (% of all CD45+ cells)
MSBC4 (% of all CD45+ cells)
1
84
0.69
1
IEA
1
1
Rectal mucosa
0.9
0.3%
0.9%
2
60
0.89
1
IEA
1
0
Gastric mucosa
1.0
1%
0.4%
3
82
0.91
1
IIEA
1
1
Gastric mucosa
2.6
0.4%
1%
4
68
0.96
1
IVA
1
2
Gastric mucosa
2.7
1%
1.2%
5
80
1.2
1
IVA
1
3
Lymph node
0.7
0.12%
