Document NYwEvga2KLbXrDy3nYajaEXw
Plasma Cell Karyotype
in Multiple
Myeloma
By James Gould, Raymond Alexanian, Angela Goodacre,
Sen Pathak, Barbara HeCht, and Bart Barlogie
Karyotypic
abnormalities
were studied in multiple myeloma
and were correlated
with clinical features.
Among 115
evaluable
patients.
46% had an abnormal
karyotype.
Tn-
somy 3. 5. 9. and 1 5 and monosomy
1 3 and 1 6 were the
most common
clonal abnormalities.
Translocations
de-
scnibed previously
in other B cell malignancies
occurred in
nine patients. including four with t(8;14)(q24;q32)
translo-
cations. The association
of all t(8;14) abnormalities
with
M ULTIPLE
MYELOMA
is a malignant
disorder of
plasma cells that secrete monocbonal immunoglobu-
bin. Cytogenetic
studies
in this disease
have been largely
unsuccessful
because of bow tumor proliferative
activity.'
Most commonly
reported abnormalities
were structural
changes of chromosomes
1 and 14, as well as a variety of
monosomies
and tnisomies.2'
` Translocations
of t(1 1 ; 14)
have also been reported,3'9"#{176} as has one patient each with
t(8;l4) and t(14;18) translocation.'2"3
This report describes
the cytogenetic
findings in a large number of patients with
plasma cell myeloma and reveals an association
of certain
chromosomal
anomalies with myeloma protein type.
METHODS
Between February 1985 and December 1986, 153 bone marrow
samples from 140 patients with unequivocal
plasma cell myeloma
were submitted
for both cytogenetic
and flow cytometnic
analysis.
Cytogenetic
studies were conducted on marrow aspirates collected in
RPMI 1640 growth medium with hepanin and colcemid (0.04
zg/mL) without mitogens. Following 20 minutes of hypotonic
treatment in 0.06 mob/L KCL, several changes of 3: 1 methanol:gla-
cial acetic acid fixative were used to fix and to eliminate RBCs.
Air-dried slides were prepared using the cell pellet resuspended
in
methanol:glaciab
acetic acid mixture (1 :1 by volume). Q-, G-, and/or
C-bandings were performed according to standard methods.'4 Iden-
tical abnormalities
in two or more cells defined a cbonal population,
except for monosomies or deletions, where three or more cells with
identical aberrations
were required'
In samples showing karyotypic
heterogeneity, ties detected.
the reported karyotype included all clonal abnonmaliA normal karyotype was confirmed when no cbonal
abnormality
was detected
among 15 metaphases
examined.
Excluded from the analysis were I 3 patients studied during remis-
sion with less than 1% monocbonal
plasma cells in the marrow on
flow cytometry and a normal karyotype; I 2 patients with less than IS
metaphases
and no clonal abnormality
were also excluded.
Flow cytometnic analyses were conducted ofceblubar DNA, RNA,
and cytoplasmic
immunoglobulin
content,'6'7 The DNA index was
defined from the ratio of fluorescence
intensities of tumor Gl/0 cells
to normal peripheral blood lymphocytes."
Tumor mass and response
to therapy were defined by standard cnitenia.'9'#{176S}tatistical compani-
sons were conducted
by chi-square
tests.
RESULTS
Of I 1 5 patients with evaluable metaphases,
46% showed
an abnormal kanyotype, which was more likely in patients
with IgA or relapsing myeloma (Table 1). The frequency of
abnormalities
was unrelated
to age, sex, tumor mass, or the
degree of marrow plasmacytosis
(Fig 1).
Chromosomal
abnormalities
were usually complex, with
multiple structural changes (translocations,
derivatives,
de-
letions) occurring in 90% and numerical deviations in 86% of
IgA protein type suggested
a pathogenetic
relationship
between
a specific karyotypic
abnormality
and myeloma
protein type. Hypodiploidy
occurred mainly in patients with
only Bence Jones protein. was associated
with resistance
to therapy. and justified the early consideration
of investi-
gational therapies.
S 1988 by Grune & Stratton,
Inc.
Table 1 . Patient Characteristics
Disease status at cytogenetic At diagnosis Unresponsive Relapsing Remission
Protein type IgG IgA Light chain only Nonsecretory
DNA ploidy Hypodiploid Diploid Hyperdiploid
study
No. Patients 115
31 44 30 10
60 30 22
3
5 25 85
% Abnormal Karyotype 46
36 45 60 (P = .12) 40
42 63(P41
0
.05)
40 40 48
patients. tnisomies
Hyperdiploid and tetnasomies
samples commonly of chromosomes
showed multiple
,3, 5, 7, 9, 1 1 15,
1 8, 1 9, and 2 1 ; monosomies
typically involved chromosomes
8, 13, 16, 20, or 22 (Fig 2). Structural
changes of chromo-
some 1 were found in 49% of patients but without a consis-
tent breakpoint or a common region of deletion (Fig 3). In
contrast,
1 3 of I 8 patients with structural
anomalies
of
chromosome
14 had a breakpoint
at q32; the remaining
five
patients showed breakpoints
at sites between q22 and q3 1
(Fig 3). One individual with a prior history of large cell
lymphoma
showed
both t(8;l4)(q24,q32)
and
t(1 l;14)(q13;q32)
in the same metaphases.
On flow cytometny
studies, 78% of the patients had
hyperdipboidy
(Table 1). There was a linear, statistical
From the University ofTexas,
M.D. Anderson Hospital & Tumor
Institute at Houston; and the Genetics Center of the Southwest
Biomedical
Research Institute, Scottsdale,
AZ.
Submitted
June 29, 1987; accepted October 8, 1987.
Supported
by Grants No. CA 37161 and CA 28771 from the
National Cancer Institute and by a grant from the Cullen Trust
Foundation.
Address reprint requests to Bart Barlogie.
MD. Professor
of
Medicine,
U. T. M.D. Anderson
Hospital
& Tumor Institute,
Department ofHematology
(Box 55). 1515 Holcombe Blvd. Hous-
ton. TX 77030. The publication
costs ofthis article were defrayed in part by page
charge payment.
This article must therefore be hereby marked
"advertisement"
in accordance
with 18 U.S.C. 1734 solely to
indicate this fact.
1 988 by Grune & Stratton. Inc.
0006-4971/88/7102-0026$3.00/0
Blood, Vol 71, No 2 (February),
1988: pp 453-456
453
454 GOULD ET AL
00 80
Inii11m
C
e-io "-20
21-30
31-40
41-50
`50
S MARROW
PLASMACYTOSIS
Fig 1 . Similar frequency of abnormal karyotype with
ing marrow plasmacytosis
defined either morphologically
abnormal
DNA/RNA
content.
0 Marrow differential;
Cytometry.
increasor by
B Flow
correlation
between DNA-derived
ploidy (DNA index) and
modal chromosome
number expressed as a karyotype index
(modal chromosome
number divided by 46; Fig 4). Among
53 patients with evaluable karyotypes,
chromosomal
hypo-
dipboidy occurred
in 1 1%, but low DNA content was
observed in only 4% (P = .01). Two patients showed concon-
dant DNA indices and karyotypes
in the tetraploid range
with some chromosome
rearrangements
present in both
copies. One patient showed a near haploid chromosome
number.
Of four patients with t(8;14)(q24;q32),
all produced IgA
myeloma protein (Table 2, Fig 5). Other translocations
associated with B cell malignancy
were also found, including
four patients with t(1 l;14)(q13;q32).
A hypodiploid
karyo-
type or a t(l 1 ;14) translocation
was present in eight of nine
patients with only light chain production.
Only three of 13
hypodiploid
patients
(23%)
responded
to chemotherapy
either prior or subsequent
to study, in comparison
with
responses in 29 of 40 other patients with abnormal cytogenet-
ics(73%,P=
.01).
No apparent
relationship
was noted between
specific
chromosomal
abnormalities
and disease manifestations,
such
as bone disease or tumor mass. In contrast to the report by
Dune et al,2' only four patients showed a deletion of the long
arm of chromosome
6, and all showed bone destruction.
Monosomy 1 3 occurred in 19 of 43 patients who had received
prior chemotherapy
but not in any of ten previously
untreated
patients (P = .02). No difference
was evident in
chromosome
number between untreated patients and those
who had prior chemotherapy.
U) C `S
a`S-
0
U) U) 0 -C
ij!..
2U --- U
-`-I
1U
U 2
II
-1i!:f'
I 8 11 14
Fig 3. Composite ideogram showing distribution
of clonal
breakpoints on the chromosomes
affected mostly with structural
aberrations. Each symbol represents one patient with a breakpoint
at this locus.
DISCUSSION
Cytogenetic
studies of myeloma have been difficult to
perform, probably due to low tumor-proliferative
activity.
Prior flow cytometnic
analyses of myeloma marrow have
revealed aneuploidy
in about 80% of patients,' suggesting
that karyotypic
abnormalities
should be identified
more
frequently
than the 30% to 50% incidence
found in this and
other studies.2"
This discrepancy
suggested
that normal
marrow cells accounted for the normal kanyotype found in
patients with cytometric
aneupboidy.
These findings high-
light the greater sensitivity of flow cytometny in assessing
ploidy in tumors with low proliferative
activity.
Among patients with an abnormal
kanyotype,
a close
relationship
was usually observed between DNA content as
determined
by cytogenetics
(modal chromosome
number)
and flow cytometry (DNA ploidy). This correlation indicates
that the identified karyotype was indeed that of the abnormal
cell population.
Hypodiploidy
was found more than twice as
frequently
by cytogenetics
than by flow cytometry.
This
discrepancy
can be attributed
to the insensitivity
of DNA
flow cytometry in detecting deletions of small chromosomes
and/or the presence of high DNA complement
despite low
[2.0
1.6
1.4
.s#{149}
SC 0
12
;s#{149}s#{149}
1.0 s-#..
0.8
Fig 2. Frequency and distribution patients with abnormal karyotype. only.
of numeric abnormalities
in
B IgA; 0 IgG; U light chain
0.6 0.8 1.0 1.2 1.4 1.6
(KARYOTYPE
INDEX
Modal No.
----,
2.0
Fig 4. Direct relationship between DNA and karyotype in myeloma patients with abnormal karyotype.
index
CYTOGENETICS IN MYELOMA
Table 2. Myeloma Karyot ype and Ig Phenotype
No. IgG IgA
Only BJP
B cell translocations (8;14)(q24;q32)' (11;14)(q13;q32)' (14;18)(q32,q21)
Hypodiploidyfl Other anomalies
4 0 4 OP= .001 4 1 12 1 1 00 13 3 4 6P= .001 34 2 1 1 2 1
`.t4. Two abnormalities
gammopathy
and one patient
were present with only BJP.
in two
patients
with IgA
455
chromosome
number.22 These findings stress the complemen-
tany power of the two techniques in tumor cell analysis, with
cytogenetics
assessing dividing cells and flow cytometry
describing the entire cell population.
This study confirms prior reports of complex numerical
and structural kanyotype abnormalities
in multiple myeboma,
especially
gains of chromosomes
3, 5, and 9; loss of chromo-
some I 3; and rearrangement
of chromosome
1 23 However,
no consistent
structural
rearrangements
were identified
other than those associated with B cell neoplasms. Transloca-
(tions 1 1 ; 1 4) have been described,3'9"0
as have single patients
with t(8;14) or t(14;18)(12,13).
In addition, important asso-
ciations with immunoglobulin
phenotype
were observed,
such as the exclusive association
of t(8;14) with IgA
myeloma protein and the prevalence
of hypodiploidy
in
patients with only Bence Jones proteinunia.
The lower
response rate among patients with chromosomal
hypodi-
ploidy was consistent
with the drug resistance
described
previously in patients with low DNA content.' The presence
of hypodiploidy
by either technique
should help identify
patients who might benefit from the early application
of
innovative
therapies,
such as high-dose
melphalan,
which has
been effective in many patients resistant to standard treat-
ment.24'25
Specific translocations
have been linked to oncogene acti-
vation in certain
human
mabignancies.262'
Thus the
t(8; 1 4)(q24;q32)
translocation,
typically associated
with
Burkitt's lymphoma, dysregulates
myc gene expression by its
juxtaposition
to the immunoglobulin
heavy chain gene.29
Fig 5. trisomies
translocation.
0-banded karyotype and t(8;14)(q24;q32).
t(8;14). is marked
of a marrow cell showing multiple A typical Burkitt's lymphoma-like by arrows.
Enchanced
myc RNA expression was present in about one
fourth of our patients with advanced myeloma studied by
Northern analysis, two of whom showed rearrangement
of
the myc gene.3#{176B}oth t(8;14) and myc gene anomalies have
occurred preferentially
with IgA myeloma. Despite seem-
ingly identical cytogenetic
aberrations
and myc involvement
in Burkitt's
lymphoma
and IgA myeloma,
molecular
differ-
ences must be postulated
to explain the different clinical
features of these B cell malignancies.
Thus different sites of
the myc gene locus may be affected, depending on whether
the translocation
occurred at an early phase of B cell
commitment
(ie, during immunoglobulin
V-D-J joining) and
leading to endemic Burkitt's lymphoma or at a later phase
(ie, during isotype switching)
and leading to sporadic
Bun-
kitt's lymphoma or IgA myeloma.3'
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