In Multiple Myeloma 14q32
Translocations are Present in
Memory B Cells but RAS Mutations
are Restricted to the Plasma Cell
Compartment
T Rasmussen, HE Johnsen, M Lodahl and M Kuehl
Background: The clonotypic
compartment in myeloma
Memory B cell
B-cell
Germinal
Center (GC)
Plasmablast
PC
Somatic mutations
The clonotypic compartment in MM
The GC B cell can differentiate into both a memory B-cell and a PC and
clonotypic cells can be identified in the memory B cell compartment in the
majority of MM patients.
Background: Genetics
· t(11;14) (Cyclin D1 High) and t(4;14) (FGFR3/IGH-
MMSET) both in 15-20% of MM.
· Cyclin D1 Low (mainly chr. 11 polysomy) in 34% of MM.
· RAS mutations, a genetic marker in the evolution of
MGUS to MM is present in 30-50% of MM.
· Increased prevalence of RAS mutations in MM-expressing
CYCLIN D1 (High and low).
Non-hyper diploid
t(11;14) and RAS
Germinal
center
Hyper diploid
B-cell
Chr.11 polysomy and RAS
MM PC
AIM
To determine if memory B-cells have the same
genetic alterations as their corresponding
malignant MM plasma cells
Memory B cell
Transformed PC
Non-malignant clonotypic remnant?
A subset of MM cases should
or
as a minimum have t(11;14)
Malignant precursor cell?
and RAS or Chr.11 polysomy
and RAS in a potential malignant
precursor.
MM PC and memory B cells
MM and MGUS PC
FACS-sorted:
CD38++/CD45-/i/CD19-/CD56+/++
CD38
RT-PCR:
Blimp-1+/XBP-1+
CD45
CD19 gate
Memory B cells
FACS-sorted:
CD38-/CD19+/CD27+
CD38
RT-PCR:
Blimp-1-/XBP-1-
Clonotypic cells identified in 70% of MM
CD27
Global RT-PCR on FACS-sorted cells
FACS:
CD38++/CD45-/i/CD19-/CD56+/++
MM PC
38DC
BM
CD45
PC sort Memory B sort
100/100
0 or 1/100
aberrant
aberrant
Global RT-PCR on FACS-sorted cells
FACS:
Global RT-PCR
CD38++/CD45-/i/CD19-/CD56+/++ mRNA
-AAAAA
MM PC
Limited RT
38
cDNA
-AAAAA
DC
300 ­ 700 bp
-TTTTT
Poly A tailing step
BM
Poly A tailing
-AAAAA
AAAAA-
-TTTTT
CD45
PCR step
Global PCR
-AAAAA-Y
X- TTTTT-
Y-AAAAA-
-TTTTT - X n
PC sort Memory B sort
PCR Primer = X-oligo(dT)-24, X = 38 bp
100/100
0 or 1/100
aberrant
aberrant
CYCLIN D1 High and Low
10
1
-actin
CYCLIN D1 high
/
0.1
D1
0.01
CYCLIN D1 low
0.001
CYCLIN
0.0001
0
102030
405060
CYCLIN D1 expression determined in FACS-sorted
CD38++CD19-CD45-/iCD56-/+/++ PC by real-time RT-PCR
Memory B cells from MM and MGUS patients
express FGFR3 or CYCLIN D1 high identical to
the autologous MM PC
Memory B
PC
L
al al
al al
CM egative orm orm M M egative M M orm orm
H
N N N
M M N
M M N N
MM
CYCLIN D1 high
0.63
0.0062 1.31
MM
FGFR3
3.78
0.0869 31.05
MGUS
CYCLIN D1 high
0.63
0.0016 0.65
IGH-MMSET+ Memory B cells
Nested IGH-MMSET RT-PCR
MM PCs
Memory B cells
500 cells
50 5
1
1
1 N
100 cells
Different numbers of MM PC
from the IGH-MMSET+ patient
were FACS-sorted into 100
50 cells
normal CD19+ cells.
1 2 3 4 5 6 7 8 N
Approx. 0.8% of memory B cells
were IGH-MMSET+
Results: Oncogenes
Patient
Oncogene
PC
Memory B
Total number of
Oncogene
Oncogene
analyzed cells
MM-1
CYCLIN D1High
1.31
0.0062
96
MM-2
CYCLIN D1High
0.16
0.0017
900
MM-3
FGFR3/IGH-MMSET
31.05
0.0869
700
MM-4
FGFR3/IGH-MMSET
12.35
0.0081
1500
MM-5
CYCLIN D1Low
0.03
-
1200
MM-6
CYCLIN D1Low/RAS
0.01
-
900
MM-7
CYCLIN D1Low
0.03
-
800
MM-8
CYCLIN D1Low/RAS
0.02
-
1200
MM-9
CYCLIN D1Low
0.01
0.0002
1200
MM-10
CYCLIN D1Low
0.009
-
800
MM-11
CYCLIN D1Low
0.008
-
700
MM-12
CYCLIN D1Low
0.005
-
800
MGUS-1
CYCLIN D1High
1.99
0.0028
1500
MGUS-2
CYCLIN D1High
0.65
0.0016
900
MGUS-3
CYCLIN D1High
0.66
-
800
MM related memory B cells are
Blimp-1 and XBP-1 negative
Memory B cells
Plasma cells
Normal Myeloma Normal Myeloma
XBP-1
Blimp-1
-actin
K-RAS61 mutations
Allele-specific competitive blocker (ACB)-PCR for
detection of K-RAS codon 61 mutations
MM PC
Memory B cells
CAA-CAC
10 N 2 2 2 N 1 1 1 1 1
RAS
Different numbers of MM PC
with a K-RAS61 CAA-CAC
CAA-CAT
mutation were FACS-sorted
into 100 normal CD19+ cells.
RAS
10 9 8 7 6 5 4 3 2 1 N P
From 6 MM patients with a K-RAS61 mutation in PC a total of 22.800
memory B cells were analyzed, without evidence of K-RAS61 mutations.
Conclusion
IGH translocations present in PC were identified in memory B cells in
4/4 MM and 2/3 MGUS cases. Thus, IGH translocation may be the
initiating oncogenic event with a mechanisms and timing of that of
normal IGH switch recombination.
The CYCLIN D1 Low phenotype is rarely observed in memory B cells.
However, the exact timing of chr. 11 polysomy remains to be resolved.
For 2 MM patients with CYCLIN D1 Low+/K-RAS61+ PCs, all
memory B cells analyzed were CYCLIN D1-/K-RAS61-.
At least for the large group of MM patients harboring RAS mutations
the clonotypic memory B-cells lacking RAS mutations cannot be
involved in maintaining the malignant clone.
Acknowledgement
Michael Kuehl
Thomas Rasmussen
Genetics Branch,
Marianne Lodahl
Center for Cancer Research,
Hans E Johnsen
National Cancer Institute,
Dept. Hematology
National Institutes of Health
Herlev Hospital
Bethesda, MD
University of Copenhagen
Denmark
Gene expression levels in PC and
memory B cells.
Gene expession level in 100 PC
Gene expression level in single cells
0.05
1.5
0.04
1
0.03
0.5
0.02
0.01
0
0
12
3
4
5
12
3
4
5
Single PC, CYCLIN D1+: 5.4 fold
Global RT-PCR: 1.7 fold
0.75
0.55
0.35
0.05
0.15
0.04
-0.05
0.03
123
45
0.02
0.01
0
Single PC, FGFR3+: 4.3 fold
1
234
5
0.005
Real-time PCR: 1.2 fold
0.004
0.003
0.002
0.001
0
12
34
5
Single memory B, CYCLIN D1+: 20.5 fold
Global RT-PCR validation
0.05
Five different PC FACS-
0.04
D1 0.03
sorts from a CYCLIN D1+
0.02
0.01
MM patient, max 1.7 fold
0
difference.
CYCLIN
123
4
5
1.5
Cyclin D1
0.035
FGFR3
0.03
Global RT-PCR
1
0.025
0.02
0.015
0.5
RT-PCR
0.01
0.005
0
0
Cyclin D1, U266
Cyclin D1, MC CAR
FGFR3, OPM-2
FGFR3,SKO007
Fold increase: 3.1
1.4