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Brief report
Translocations involving the immunoglobulin heavy-chain locus are possible
early genetic events in patients with primary systemic amyloidosis
Suzanne R. Hayman, Richard J. Bailey, Syed M. Jalal, Gregory J. Ahmann, Angela Dispenzieri, Morie A. Gertz, Philip R. Greipp,
Robert A. Kyle, Martha Q. Lacy, S. Vincent Rajkumar, Thomas E. Witzig, John A. Lust, and Rafael Fonseca
Primary systemic amyloidosis (AL) is a
plasma cell (PC) dyscrasia with clinical
similarities to multiple myeloma (MM) and
monoclonal gammopathy of undetermined significance (MGUS), but its molecular basis is poorly understood. Translocations at the immunoglobulin heavychain (IgH) locus, 14q32, are likely early
genetic events in both MM and MGUS and
involve several nonrandom, recurrent,
partner chromosomes such as 11q13,
16q23, and 4p16.3. Given the similarities
between MM, MGUS, and AL, bone marrow clonal PCs were evaluated in 29 patients with AL using interphase fluorescence in situ hybridization (FISH)
combined with immunofluorescence detection of the cytoplasmic light-chain (cIgFISH) for the presence of 14q32 translocations and the t(11;14)(q13;q32). Of 29
patients studied, 21 (72.4%) showed results compatible with the presence of a
14q32 translocation, and 16 (76.2%) of
those had translocation (11;14)(q13;q32)
for an overall prevalence of the abnormality of 55%. IgH translocations are common
in AL, especially the t(11;14)(q13;q32).
(Blood. 2001;98:2266-2268)
© 2001 by The American Society of Hematology
Introduction
Amyloidosis (AL) is a plasma cell (PC) dyscrasia, similar to
multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS),1,2 characterized by the presence of
clonal PCs in the bone marrow (BM) and extracellular deposition
of amyloidogenic light chains with resultant organ dysfunction and
an overall median survival of 1.5 years.1,2 The usually fatal
outcome of the disease does not permit long-term observations of
clonal growth.
Chromosomal translocations involving the immunoglobulin
heavy-chain (IgH) locus may be initiating events in the development of several B-cell malignancies,3,4 including MGUS and MM.5
IgH translocations in MM involve a number of recurrent, nonrandom partner chromosomes including 11q13 and 4p16.3.14-16 Using
the cytoplasmic light-chain (cIg)–fluorescence in situ hybridization
(FISH) technique, we examined 29 patients with AL for the
presence of IgH translocations, including t(11;14)(q13;q32).
Study design
Patient samples
Twenty-nine patients (10 women, 19 men; median age, 62 years) were
studied. Institutional review board approval was obtained, and all patients
gave written, informed consent for a research BM sample to be collected at
the time of routine clinical procurement. Diagnostic criteria for AL included
histologic proof of amyloid deposition in addition to the presence of a
monoclonal protein or demonstration of monoclonal PCs. Five of the 29
patients had previously been treated with alkylating agents. None of the
patients had clinical evidence of MM.
From the Departments of Hematology and Internal Medicine and of Laboratory
Medicine and Pathology, Mayo Clinic, Rochester, MN.
Submitted January 8, 2001; accepted May 17, 2001.
Supported in part by Public Health Service grant R01 CA83724-01 from the
National Cancer Institute. P.R.G. is supported in part by research grant P01
CA62242 from the National Cancer Institute and by ECOG grant CA21115-25C
from the National Cancer Institute. R.F. is a Leukemia and Lymphoma Society
Translational Research Awardee. R.F. and P.R.G. are also supported by
the Mayo Foundation and the CI-5 Cancer Research Fund, Lilly Clinical
2266
Cell enrichment and cIg-FISH
PCs were selected with CD 138 magnetic microbeads (Miltenyi Biotec,
Sunnyvale, CA) after enrichment for mononuclear cells through centrifugation on a Ficoll-Hypaque gradient, density 1.076 (Pharmacia-Biotech,
Uppsala, Sweden). Cytospin preparations were prepared according to a
slight modification of our previously published cIg-FISH technique.6 We
scored 100 PCs in all but 2 cases. One repeatedly failed to hybridize; in the
other, we could only score 50 cells for t(11;14)(q13;q32), respectively. Each
sample was scored by 2 independent examiners who had a high degree of
agreement (r 2 ⫽ 0.795).
FISH probes
Break-apart strategy. Probes that hybridize to the variable (VH; cosmid
clone) and constant regions (CH; BAC clone) of the IgH locus7 were directly
labeled with SpectrumRed and SpectrumGreen, respectively (Vysis, Downers Grove, IL). The presence of a 14q32 translocation was demonstrated by
segregation of the signals (break-apart strategy; Figure 1). A normal pattern
consisted of 2 pairs of closely associated red and green signals. An
abnormal pattern was defined as a distance greater than 3 signal widths
between probe pairs. An equivocal pattern was one in which there was loss of a
probe signal (1 red-green pair/1 red or 1 red-green pair/1 green), representing
deletion, an unbalanced translocation, or technical inability to discern the
presence of a signal because of small size or background staining.
Fusion strategy. For the detection of the t(11;14)(q13;q32), on separate
experiments, we used the same IgH probes (labeled with SpectrumGreen)
and a pool of cosmid and P1 clones encompassing all reported breakpoints
in human MM cell lines with the t(11;14)(q13;q32)8-10 (labeled with
SpectrumRed). The normal pattern was that of 2 pairs of nonassociated
signals (2 red/2 green). Fusion of 2 signals in a cell was considered
indicative of the translocation.
Investigator Award of the Damon Runyon-Walter Winchell Foundation.
Reprints: Rafael Fonseca, Department of Hematology and Internal Medicine,
200 First St SW, Bldg W10B, Mayo Clinic, Rochester, MN 55905; e-mail:
[email protected].
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2001 by The American Society of Hematology
BLOOD, 1 OCTOBER 2001 䡠 VOLUME 98, NUMBER 7
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BLOOD, 1 OCTOBER 2001 䡠 VOLUME 98, NUMBER 7
IgH TRANSLOCATIONS IN PRIMARY AMYLOIDOSIS
2267
Figure 1. Normal and abnormal patterns of hybridization with both strategies. (A) Hybridization of the VH
and CH probes to the correct location at band 14q32 on
normal human male metaphases. (B) Normal pattern for
the VHCH probes with 2 pairs of closely associated
signals. Note the intense blue fluorescence of the cytoplasmic light chain. (C) Cellular signal pattern indicative of a
14q32 translocation. The segregation of a pair of signals
(break-apart) in a large percentage of cells allows for the
identification of a patient with an IgH translocation.
(D) Hybridization of the 14q32 and 11q13 probes to the
correct locations on chromosomes 14 and 11, respectively, on normal human male metaphases. (E) Normal
pattern for the 14q32/11q13 fusion strategy. There are no
fusion signals observed, and there are 2 pairs of nonassociated green (14q32) and red (11q13) signals. (F) Abnormal comigration (fusion) of signals indicative of a t(11;
14)(q13;q32). Because of the small signal size arising
from the VH portion of the IgH probe (owing to the V/D/J
recombination and DNA excision), the pattern most frequently observed is presumed to be that of fusions in the
der14 and less commonly in the der11.
Statistical considerations
The normal range of PC patterns was documented previously using cIg-negative
cells derived from patient samples and polyclonal PCs from BM donors. A
patient was said to have an IgH translocation if the percentage of PCs exceeded
the mean ⫾ 3 SD.11 The upper limit of normal for the break-apart and fusion
strategies was less than 10% for both. We arbitrarily chose an upper limit of
normal of 25% for greater specificity.
Table 1. Patient clinical and laboratory features and associated FISH results
No.
Sex
Age
IgH status
BM PC
(%)
␬:␭ ratio
Heavy
chain
Serum M
spike
(g/dL)
Urine M
spike
(g/24 h)
VH/CH
Pattern
t(11;14)(q13;q32)
Positive
(%)
Equivocal
(%)
Positive and
equivocal
Pattern
Positive
(%)
Newly diagnosed patients
1
M
63
t(11;14)
19
⬍ 1:99
ND
IF
1.5
1F1R1G
89
11
100
2F1G2R
100
2
F
78
t(11;14)
8
1:40
IgG
0.6
0.9
1F1R1G
84
8
92
1F1G2R
86
3
F
51
t(11;14)
33
⬍ 1:99
ND
IF
IF
1F1R1G
79
8
87
1F1G2R
4
F
62
t(11;14)
12
1:2
IgG
0.4
IF
1F1R1G
75
6
81
1F2R
5
M
71
t(11;14)
4
1:4
ND
ND
IF
1F1R1G
66
10
76
1F1R1G
68
6
M
62
t(11;14)
13
1:54
IgG
0.8
ND
1F1R1G
57
30
87
1F1R1G
95
7
M
53
t(11;14)
22
⬎ 99:1
ND
ND
IF
1F1R1G
55
31
86
1F1G2R
85
8
M
57
t(11;14)
26
⬎ 99:1
IgA
IF
IF
1F1R1G
54
36
90
1F2R
85
89
100
9
M
77
t(11;14)
1
1:1
IgG
ND
0.7 (␭)
1F1R1G
53
19
72
1F1G1R
71
10
M
65
t(11;14)
10
1:4
ND
ND
ND
1F1R1G
38
30
68
1F1G1R
95
11
M
63
t(11;14)
3
93
12
M
75
t(11;14)
25
13
M
40
t(11;14)
14
M
41
15
M
74
16
F
67
17
F
57
⬍ 1:99
⬎ 99:1
ND
IF
3
1F1R1G
30
59
89
1F1G3R
IgG
1.6
1.5
1F1R1G
28
68
96
1F2R1G
72
ND
IF
IF
1F1G
21
69
90
1F2R
68
8.8
1:44
t(11;14)
5
1:25
ND
ND
0.5
1F1G
19
66
85
1F2R1G
96
14q32⫹
10
1:60
IgA
0.7
1.2
1F1R1G
41
35
76
2G2R
15
14q32⫹
1
1:1
ND
IF
IF (␭)
1F1R1G
38
14
52
2G2R
19
14q32⫹
1
⬍ 1:99
ND
ND
IF
1F1R1G
27
17
44
2G2R
18
18
F
58
Equivocal*
3
19
M
67
Negative
4.6
20
M
64
Negative
9
21
F
37
Negative
15
22
M
65
Negative†
13
23
M
60
Negative
38
24
F
70
Negative
34
1:13
IgG
1.7
IF
2F1G
7
83
90
2G2R
10
⬍ 1:99
IgA
ND
IF
2F
18
14
32
Fail
—
1:23
IgG
0.9
IF
2F
9
18
27
2G2R
16
ND
ND
IF
2F
23
19
42
2G2R
15
ND
ND
ND
2F
20
20
40
Fail
—
IgA
2.5
ND
2F
19
24
43
2G2R
13
IgA
1.2
IF
2F
14
11
25
2G2R
23
72
18:1
1:13
⬎ 99:1
⬍ 1:99
Patients with previous alkylator chemotherapy
25
M
74
t(11;14)
5
4:1
ND
ND
ND
1F1G
25
65
90
1F1G2R
26
F
45
t(11;14)
17
⬍ 1:99
IgG
IF
0.05
2F1G
12
64
76
1F1G2R
80
27
F
55
14q32⫹
3
⬍ 1:99
IgA
0.5
ND
1F1R1G
75
16
91
2G2R
15
28
M
54
Negative
10
1:1
IgM
0.8
0.07 (␭)
2F
15
19
34
2G2R
23
29
M
54
Negative‡
22
⬎ 99:1
IgG
1
0.2
2F
7
9
16
Fail
—
F indicates fusion; G, green; R, red; 14q32⫹, IgH translocation without partner identified; IF, immunofixation; ND, not detected.
*Equivocal for 14q32 and negative for t(11;14).
†Patient with monosomy of chromosome 14 in 55% of PCs.
‡Patient with monosomy of chromosome 14 in 76% of PCs.
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2268
BLOOD, 1 OCTOBER 2001 䡠 VOLUME 98, NUMBER 7
HAYMAN et al
Results and discussion
Break-apart strategy (VH/CH probes)
Of these 29 patients, 16 (55%) had definitive evidence of an IgH
translocation using the VH/CH strategy (Table 1). The median percentage
of abnormal PCs (MAPC) was 55% (range, 27%-89%). Additionally, 5 (17%) patients had an equivocal pattern compatible with a
possible IgH translocation (MAPC, 66%; range, 64%-83%). In
total, 21 of 29 (72%) patients had evidence of an IgH translocation
by the VH/CH probe patterns.
t(11;14)(q13;q32)
Twelve of 16 (75%) patients with definitive IgH translocations by
the VH/CH strategy had a t(11;14)(q13;q32) (MAPC, 87.5%; range,
68%-100%). In addition, 4 of the 5 (80%) patients with an
equivocal pattern using the VH/CH strategy also had a t(11;14)(q13;
q32) (MAPC, 76%; range, 68%-96%). Altogether, 16 of the 29
(55%) patients had evidence of a t(11;14)(q13;q32), accounting for
76% of all IgH translocations detected (16 of 21 patients). There
was no statistically significant difference in the BM percentage PC
between patients with and without the abnormality detected by
either strategy (Wilcoxon signed-rank test, P ⬎ .2)
Other
Four patients had definitive evidence of an IgH translocation by the
break-apart strategy that did not involve the 11q13 locus (MAPC,
64%; range, 44%-91%). One patient had a possible IgH translocation with an equivocal pattern by the VH/CH in 90% of PCs (not
involving 11q13). Eight patients did not have evidence of IgH
translocations, and 2 of those patients had only 1 pair of VH/CH
probes in 55% and 76% of their cells, respectively, reflecting
monosomy 14 or deletion of 14q32.
We have shown here that most patients with AL harbor IgH
translocations in clonal PCs. In AL, the deposition of monoclonal
light chains is the cause of the sickness and death associated with
the disease, whereas clonal progression is usually not observed.12
Our results support the hypothesis that AL is the same disease
entity as MGUS/SMM (at the clonal level), with the clinical
differences (phenotype) arising from the amyloidogenic potential
of the monoclonal light chain. Most patients with AL have a low
degree of BM involvement and rarely have overt MM.13 We
speculate the occasional patient with AL (approximately 5%), who
also has coexistent MM, may have a lesser amyloidogenic variant
of the light chain or a more rapid clonal progression.14 The
frequency of t(11;14)(q13;q32) is much greater in AL than has been
noted for MM15 or MGUS16-18 in some series but not in others.19
Several facts allow us to conclude that the prevalence of the
t(11;14)(q13;q32) we report in AL is correct. We used 2 independent scorers, and both concurred in the scoring of abnormal PCs in
each sample with a high degree of agreement. The percentages
of abnormal PCs by the break-apart and fusion strategies were
similar in each patient, and the abnormalities were usually seen in
most of the clonal PC. Finally, we also observed a higher
proportion of t(11;14)(q13;q32) in patients with MGUS than in
patients with MM.19 The frequencies of other partner chromosomes
are being studied to assess the pathogenetic potential of these
abnormalities, and we speculate that other partner chromosomes
will also be involved.20-22
The results of our study identify cytogenetic lesions in the
clonal PCs of patients with AL, similar to those observed in MGUS
and MM. In addition, previous work has shown that VL gene
sequences are highly somatically mutated, suggesting commonality
among these dyscrasias.23 We have previously shown that aneuploidy is equally present in AL, MGUS, smoldering MM, and
MM.24 Hallek3 proposes a step-wise transformation model for the
development of MM from MGUS, and we propose the inclusion of
AL in the model where AL is similar to MGUS and smoldering
MM, albeit with an “unlucky” protein.
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From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
2001 98: 2266-2268
doi:10.1182/blood.V98.7.2266
Translocations involving the immunoglobulin heavy-chain locus are possible
early genetic events in patients with primary systemic amyloidosis
Suzanne R. Hayman, Richard J. Bailey, Syed M. Jalal, Gregory J. Ahmann, Angela Dispenzieri, Morie A.
Gertz, Philip R. Greipp, Robert A. Kyle, Martha Q. Lacy, S. Vincent Rajkumar, Thomas E. Witzig, John A. Lust
and Rafael Fonseca
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