Establishment of an Amyloid Forming
Human Cell Line
Bi
Bonnie K. A
d
ren t
dt
Gregory J. Ahmann
Rafael Fonseca
Jonathon Keats
Erin M. Mulvihill
Angela Dispenzieri
Laura A.
A. Sikkink
Sikkink
Marina Ramirez-Alvarado
Steven R. Zeldenrust
Xiaosheng Wu
Diane F. Jelinek

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Amyloidosis
Light Chain
Unfolding
Misfolding
(folded)

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Patient History Timeline
Diagnosis:
Diagnosis: AL Amyloidosis
MM with AL
ALMC-2
Amyloidosis
established
Sept
Oct
Nov
Dec
Jan
Feb
Mar
Apr
2005
2005
2005
2005
2006
2006
2006
2006
ALMC-1
Patient deceased
Autologous
established
PBSCT

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Amyloid Deposition in BM Aspirates
Oct 2005
Mar 2006

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ALMC-1/ALMC-2 Ig Gene Usage
bda
C-1
trol
C-2
t
m
M
M
La
con
AL
1:3
AL
1:3
Heavy Chain: VH 3-21 (9.7% mutation)
FLC-
DH 2-21
JH 4b
C region: IgG1
140
ALMC-1
120
Light chain: V 6-57*01 (3.3% mutation)
100
Nil
J 3*02
g/mlµ 80
IL 6
-
C region: IgC3*03
60
IGF-I
40
IL-6+IGF-I
FLC- 20
0

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Phenotypic Comparison of AL Cell Lines
ALMC-1
ALMC-2
30
25
20
15
MFI 10
5
0
1a
17
CD2
CD3
CD5
CD10
D1
CD14
CD16
CD19
CD20
CD22
CD25
CD27
CD38
CD40
CD44
CD45
D49d
D49e
CD52
CD86
D1
D126
D138
D154
C
CD28
CD56
CD73
CD80
C
C
C
C
C
W130
C
D
C
C
D
C
C

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Cytokine Responsiveness
33
ALMC-1
26
ALMC-2
19
creasen ynthesis)
In
S
12
Fold
(DNA
5
-2

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Genomic Abnormalities of AL Cell Lines
Source
Diagnosis
ALMC-1
pre-PBSCT
ALMC-2
MYC Amp
25%
100%
100%
100%
CEP3
Normal
Tetraploid
Normal
Normal
CEP7
Normal
Tetraploid
Normal
Normal
CEP8
Normal
Tetraploid
Normal
Normal
CEP11
Normal
Tetraploid
Normal
Normal
CEP15
Normal
Tetraploid
Normal
Normal
CEP17/p53
40% del p53
100% del p53
100% del p53
100% del p53
RB/319
del 13q
del 13q
del 13q
del 13q
11;14
Negative
ND
ND
ND
c-MYC
4;14
ND
ND
Negative
ND
14;16
Negative
Negative
ND
Negative
ND=Not Done

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High Resolution aCGH
Chromosome 3
Chromosome 8
Chromosome 22

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Fibril Formation from Purified Light Chain
AL
ence
67500
MC-1
65000
62500
60000
luoresc
(RFU)
F
m 30000
n 20000
10000
485
A
mission
at
L
0
MC-2
1X ThT ALMC-1 1X ThT ALMC-2
E
ThT

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Amyloid Mouse Model
ALMC-2
Non-irradiated NOD-Scid
10x106

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Anal sis
y
of
of Imm
Immunoglob lin
u
E p
x ression
pression
in Excised NOD/SCID Tumor Tissue
A. Ig RNA Expression
B. Ig Protein Expression
/DAPI
ALMC-1/2
G
1/2
Specific
cIg
IgVH primers
/DAPI
-actin
c

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Phenotypic Analysis of
of Excised
Excised
NOD/SCID Tumor Tissue
CD38
gp80
CD19
CD86
CD19
gp130
CD138
CD45
CD20
CD28

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Amyloid Formation and Organ
Deposition
MR
Mouse i
Ri h
g t
ht Kid
Kidney
Mouse Left Kidney

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Conclusions
Successful establishment of cell lines (pre and
post PBSCT) from a patient with primary
amyloidosis
Cell line-derived (ALMC-1 and ALMC-2) purified light
chain formed amyloid fibrils in vitro
Successful tumor formation of cell lines in
NOD/SCID mice
Unprecedented amyloid formation and
deposition of
of human free light chain in
in
cell line-injected mice
Invaluable tool and resource to better understand
amyloid producing
producing plasma
plasma cells and AL
AL amyloidosis
amyloidosis

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