cell involvement Phenotypic heterogeneity in

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1988 71: 861-865

Phenotypic heterogeneity in aneuploid multiple myeloma indicates pre-B cell involvement J Epstein, B Barlogie, J Katzmann and R Alexanian

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Phenotypic

Heterogeneity

in Aneuploid

Indicates By Joshua

The expression

of early

fl2-microglobulin by

aneuploid

from

44

patients

correlated

tamed cell

antigen

cells

of

as

well

ULTIPLE

bone

in 1 7%,

90%

and

of the

the

surface

84

B2M;

in 23%

MYELOMA

is a B cell

related from

reported

more

globulin

(B2M).2

cells has concordant

The

from

the

serum

neoplastic

.

level

isotype

lymphoid myeloma.35

expression

clinical

with CALLA+ Our laboratory

of earlier

course

has

tive assessment of relationship to DNA tumor

cell

been

the blood lymphocytes

bone

marker

marrow

myeloma

present

other which in about

44 patients

MATERIALS

features

cellular markers in serves as an unequivof patients

We now report our mature B cell markers

from

an

in myeloma cellular

80%

AND

with

with

studies on the by tumor cells

aneuploid

myelo-

METHODS

Bone marrow aspirates were obtained from 44 patients with DNA-aneuploid myeloma in various stages of disease; ten of the patients were previously untreated. Informed consent was obtained from the patients before bone marrow aspiration. After FicollHypaque separation (density, 1,077 g/mL), interphase cells were washed in phosphate-buffered saline and reacted with a panel of monoclonal antibodies to surface antigens by using either direct or indirect immunofluorescence assays. After immunostaining, the cells were fixed in ice-cold 70% ethanol and counterstained with propidium iodide at 20 zg/mL for correlated DNA phenotypic analysis, which was carried out with an EPICS V flow cytometer (Coulter Electronics, Hialeah, FL).9 The investigated surface markers included the pre-B cell antigen CALLA, which was studied by using the monoclonal antibody iS’#{176}; B2, which is expressed by early to mature nonactivated B cells”; B4, which is expressed by all B cells but not by mature plasma cells’2”3 (Coulter); and the plasma cell marker RI-3, which was kindly provided by Dr iA. Katzmann (Mayo Clinic, Rochester, MN).5”4 In addition, cytoplasmic immunoglobulin (clg) was also analyzed by using both anti-K and anti-A

Blood, Vol 71, No 4 (April),

1988:

pp 861-865

CALiA

a novel

and

clg

myeloma

in the

normal

in

46%

of

phenotype

all

patients

without

differentiation

known

of B cells.

CAiiA

and clg were independently expressed and gave rise to CAiiA+ /clg-, CAiiA+ /clg+, and CAiiA/clg+ cells. The association of CALiA and mature plasma cell markers may define discrete stages of neoplastic plasma cell differentiation. a 1988 by Grune & Stratton, Inc.

light chain antisera [F(ab’)2 fragments; Cappel Laboratories, West Chester, PA].’5 We also studied surface expression of B2M (Dako, Santa Barbara, CA). which is secreted in proportion to tumor mass.2 Separate aliquots of all samples were also subjected to acridine orange staining for correlated analysis 01 DNA and RNA content as previously described.’6 Routinely, 10,000 cells were analyzed for each marker. Quantitative analysis was carried out with a Terak computer (Scottsdale, AZ) using cells stained with nonreactive antibodies as matching controls. RESULTS

An example

in vitro immuno-

Recently,

described

of

Alexanian

of

the biology and the clinical were devefoped for quantita-

B cell and aneuploidy,

plasma cell myeloma.8 expression of early and in the ma.

secretion.6

plasma cells.7 has investigated

B

of idiotypeleukemia anti-

cells in CALLA+

and

as a means of understanding course of the disease. Methods

ocal

typi-

of fl2-micro-

from blood and bone marrow could be differentiated to mature plasma cells with concordant monoclonal aggressive

in

cell morphology Patient survival

involvement

Raymond

studied.

malignancy

suggested by the presence common acute lymphoblastic

gen (CALLA)-positive patients with multiple

globulin

acute

to the tumor mass at diagnosis, which can a number of laboratory parameters’ or, as

recently,

been and

con-

plasma

present

samples

cally associated with mature plasma monoclonal immunoglobulin secretion

is inversely be estimated

patients

was

of

by

common

(CAiiA)

and

counterpart

cytometry.

mature

Katzmann,

identified

evaluated

Myeloma

Involvement

Coexpression

aspirates

was

expressed as

surface

marrow flow

antigen

Jerry

immunoglobulin

myeloma

almost

clg and

leukemia

M

in

Cell

Barlogie,

B cell markers,

cytoplasmic

multiple

cells

Ri -3

B2

Bart

immunofluorescence

tumor

55%,

and

with

monoclonal

lymphoblastic

and

tumor

DNA

Myeloma

and mature

(B2M)

(clg)

Epstein,

Pre-B

Multiple

of DNA

a hyperdiploid tumor with

phenotype

myeloma

analysis

is presented

cells showed a high RNA anti-B2M, J5, and R1-3;

monoclonal compartment

light chain had a low

K

for B2M complete

65% of the cases. aneuploid tumor

Monoclonal cells of

respectively.

pre-B

clg

positively cIgA with

Because of scarcity could be performed

antigen

tumor (Table

aneuploid monoclonal

was and

cell clg, of in

and R I -3 were present in and 89% of the patients,

88%

cell

cells Rl-3,

content; reacted and contained

and iS. studies

by aneuploid myeloma studied for this marker tumor clg,

with

1 . 1-lyperdiploid

reactivity. Cells in the diploid RNA content, did not express

but were positive marrow samples,

The

in a patient

in Fig

CALLA

was

expressed

cells in 55% of 43 samples 1). Phenotype coexpression by

observed B2M, and

in 80% in 45%

of patients for of the patients,

the pre-B cell antigen CALLA was present together with monoclonal clg and the mature plasma cell antigen Rl-3 (Fig I and Table 2). Only a few patients (13% and 15%, respectively) demonstrated coexpression of CALLA with B2 and

B4

From tem

antigens.

the

Department

Cancer

Houston,

There

Center,

Supported

in part

16672from Address

reprint and

Houston,

TX

to Joshua 15/5

Sys-

Institute.

MN. 9, 1987.

37161.

ofHealth,

Institute.

Tumor

Epstein,

CA

28771.

and

CA

Bethesda,

MD.

DSc.

Anderson

Holcombe

M.D. Blvd.

Box

55.

77030.

“advertisement” 1988

CA

the

of Texas

and

November

No.

between

University

Rochester,

Institutes

requests

payment.

indicate

correlation

Hospital

accepted

by Grants

Tumor

The publication

©

Clinic,

29. 1987;

the National

Hospital

charge

Anderson

and the Mayo July

no

of Hematology.

M.D.

Submitted

was

costs

ofthis

article

This

article

must

in accordance

with

were defrayed therefore

18 U.S.C.

in part

be hereby §1734

by page marked solely

to

this fact. by Grune

& Stratton.

Inc.

0006-4971/88/7104-0002$3.00/0

861


EPSTEIN

862

ET AL

r..’”.’”..’.”

B2M

(

1”’’

f1

a.

R1-3

C 0

‘S

a

“S

K

I”

a 0 C C 1S

S S

CIgA

S

S.

a.

S.

a.

CIgA

I

‘S

is

.(

,‘

.

a.

4#{149}

“S

S.

S.

-

4

S

-

Cellular

-,

DNA

S.

S.

IS

a.

am

‘.S

Content

Fig 1 . Phenotypic heterogeneity in a patient with hyperdiploid multiple myeloma. Hyperdiploid cells show a high RNA content; with anti-B2M. J5. and Ri -3; and show monoclonal clgA K expression. Diploid cells have low RNA contents without monoclonal reactivity but express B2M and CALLA. Abscissa. DNA content; ordinate, fluorescence intensity of cellular feature.

proportions

of

tumor

cells

B2M,

or clg

that

stained

CALLA,

R1-3,

positive

for these

expression patients

in plasma cell myeloma (Fig displayed an additional diploid

line

on clg

presented content and

analysis, in

and

markers,

Fig

thus

3.

R 1-3; diploid

whose

indicating

a representative

their

cells

clg

positive

cells

also

contained

a low RNA content in all patients with

cells, the diploid diploid cells were

cells only

of which

with clg

positive, but had R1-3. Interestingly,

were

is

a high

RNA

for CALLA

(iS)

K,

were

and did CALLA+

cells.

revealed

Further three

CALLA+/clg+, and alone or in combination,

aggressive The

course

median

with

survival

1 . CAiLA

Expression

Myeloma

in Aneuploid

Table

2.

Percentage

of patients of positive

studied patients

CALLA-/clg+, at times with

expres-

CALLA+/clg-, that different

are present ploidy levels

(Figs 3 and 4). It has been myeloma has a particularly

a short from

Coexpression

Aneuploid

median

survival

diagnosis

Tumor

of Early

Myeloma

CALLA+ CALLA+

Plasma

Positive

Cells

B4 CALLA

Number

of CALLA/clg

phenotypes,

(ie, diploid and hyperdiploid) reported that CALLA-positive

CALLA

Table

examination

different

of

of 6 months.7

2 1 patients

with

CALLAnot express aneuploid

also expressed CALLA. seen in association with

aneuploid sion

independent

example

x were

for

cells

2). Seven of the 44 tumor DNA stem-

Hyperdiploid

monoclonal

positively

in patients

react clg

B4

CALLA

B2

R1-3

82M

clg

35

43

33

40

44

44

R1-3

23

55

17

89

90

88

B2M

B2

15

and Late Cells

B Cell Markers

(Percentage

by

of

Patients)

B2

R1-3

B2M

clg

4

19

19

14

13

44

45

46

17

20

78

81

13

79


CALLA/cIG

CO-EXPRESSION

BY

MYELOMA

TUMOR

CELLS

863

100

100

Med

11,2

A,

80

A

80

60

60

A 40

M,d

.

.

‘

20

.

A,

.. .

Med

20

0

20

40

60

80

100

0

20

40

CALLA

60

80

100

CALLA

0 100

M.d

100 M.d

U

A,

A,

80

80

60

60 A

40

of

23

mycloma

patients

months;

P

with .004)

was significantly

CALLA-negative

(Fig

Expression bone

of

and marrow

of 44 patients

with

myeloma by using correlated flow cytometry. Tumor cells DNA

aneuploidy,

myeloma patients

which

patients.8 reacted

The

longer

than

that

(>80

24

v

with

the

B

was evaluated

DNA-aneuploid

cell

in the

tumor mature

in more cells

than

of about

plasma

cell

60

expressed

80

0

100

20

40

80% 90%

marker

by of all of

the

Rl-3,

surface

B2M,

a unique tumor In casesofconcurrent

these

markers

and represented CALLA +/clg-, Although showed

iS

80

100

POSITIVE

cell features R I -3 and et al.’7 Coexpression counterpart in normal sent

60 81-3

and

however, tumor cells ofone pre-B cell antigen CALLA

multiple

DNA and immunofluorescence are unequivocally identified

is present

40

PERCENT

disease

cells

20

81-3

differentiation-associated

B2M by tumor

20

0

5).

several

surface

Med

.. .

20

DISCUSSION

markers

.‘

40

S

Fig 2. Independent expression of cellular markers by myeloma tumor cells. The percentages of cells positive for each marker in bone marrow samples coexpressing these markers are plotted against each other. The correlation coefficient for each plot was < .5.

CALLA-positive

A,

M.d

were

clg.

cell

phenotype CALLA

expressed

the

Unexpectedly.

patients expressed the with the mature plasma

clg, recently also of CALLA and B cell differentiation

tumor cell CALLA

none of reactivity

contained

halfofthe together

reported by Grogan clg has no known and could repre-

in myeloma. and clgor Rl-3 independently

subpopulations +/clg+ . or

CALLA-negative in DNA-diploid

reactivity,

of each

other

that were either CALLA -/clg+.

cells,

aneuploid all cases

cases with

C

0

0, 0, 0,

w 0,

0.

2 0,

60

40

Cellular Fig 3. expressed

Phenotypic heterogeneity CALLA (J5). Hyperdiploid

DNA

in a biclonal myeloma with two DNA cells also reacted with Ri -3. whereas

Content stemlines. Both diploid diploid cell expressed

and hyperdiploid surface B2M.

cells contained

clg and


864

ET AL

EPSTEIN

.

II

---

r-’-

r

CALLA

‘

-

(

-.

a.

a.

“S

“S

a.

a.

C,)

as

55

Ui

S.

z

.

-

-

-

‘

-

-

-

,

-

-

- .

-

-

-

.

-

-

-

..

50

z Ui 0

o.--

--;

-.:o---ai-

a.t:Ss

1#{149}

a.

S

.::tr

--‘::

S

..

z

Ui

0

Cig

Ui

0

-

by

of this

pre-B

but usually ity

with

without the

-

aneuploid cell

tumor

marker

and the additional presence compartment may reflect

CALLA-/clg+

I

a.

JF4SIfJ_!

6.5

as

-

a.

I.

45

.

CONTENT

in aneuploid indicates the

myeloma. presence

cells

also

phenotypic

aneuploid

tumor

mature ofa

showed cells,

cell

diploid

CALLA+/clg-

a.

lation in all suggests the more

the

precurand then CALLA-/ cell

popu-

-

subtle

in aneuploid of a diploid

DNA

aneuploidy

genetic

changes

Such

CS

CALLA+ myelomas tumor progenitor cell

may

therefore

associated

a model

could

be distal

to

with

malignant

the

emergence,

explain

(eg, hypodiploidy advances’8 and

in a would

imply result

that clonal changes during the course of the disease from adaptation in response to selective pressures

exerted The

by chemotherapy concept of pre-B

genetic t(l

l;14)]

and

given at subcurative dose. cell involvement in the development

is supported

studies: were

c-myc

by cytogenetic

lymphomalike found in patients

and

reported.#{176}2’

Bc/I

These

receptor plasma malignant proximal

as well

have suggest

myeloma recently

‘y gene rearrangements cell myeloma22 also raises transformation

[t(8;14) and myeloma’9;

also

been

that occurs reported

the

recently malignant

at an early stage finding of T cell

in three patients with the possibility that the

in multiple

to the commitment

as molecular

translocations with multiple

involvement findings

transformation in multiple of B cell maturation. The

0 0. 0 0.

;S

(a) CALLA - - /clg - + ; (b) of marker for the diploid and

in some patients, of new ploidy levels hyperdiploid myeloma) as the disease

of myeloma

C 0

#{243}s is

S

were observed: . the absence

and only existence

transformation.

compart-

S

Three phenotypes of cellular marker;

compartment.

in

a.

is

i;.

heterogenecell

phenotype

is

CALLA+/

of a CALLA+/clgdiscrete stages

plasma

as

DNA

development ofmultiple myeloma: CALLA+/clgsor cells mature progressively to CALLA+/clg+ presence

-‘

as

of CALLA+/clg-,

and

expected

Lai-

a.

by DNA-diploid

clg or RI -3. This

ments diploid

The

1

-

-.

.

clg+,

to the

‘

-

-

coexistence

clg+.

-

‘C

a. -

Fig 4. Heterogeneity of CALLA expression CALLA + + /clg - + / (c) CALLA + + /clg + 4- . + hyperdiploid cells. respectively.

expression

-

-

..

C.-

expression

-

-

i: _,#1.#{149}$

CALLA

-

‘C -

-

‘

,

I.-...-.-

-J LA.

r

I.

es

myeloma

to lymphoid

occurs

lineage

even

differentia-

tion. expression

CALLA

by myeloma

association with an aggressive Even though obtained from their diseases, in fact, seem 0

10

20

30

40

50

60

70

80

90

Months Fig 5. pared with

Superior CALLA

-

survival patients.

of CALLA

+

myeloma

patients

com-

CALLA

our results to indicate

+ myeloma,

cells

course patients

has

been

and short at different

noted

in

survival.7 stages of

are at variance with this report and, a more favorable clinical course in

perhaps

as a result

therapy (vincristine, Adriamycin, marked efficacy also in CALLA-positive tic leukemia in adults.23

of different

chemo-

dexamethasone), with acute lymphoblas-


CALLA/cIG

CO-EXPRESSION

BY

MYELOMA

TUMOR

865

CELLS

ACKNOWLEDGMENT

The authors wish to express Teague for her excellent technical her secretarial assistance.

I

their most assistance

sincere thanks to Kim and to Eva Menefee for

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