Immunoglobulin kappa and lambda light chain dual genotype rearrangement in a patient with kappa-secreting B-CLL

0277-537918893 oo+o.oJl Prrqamon Prrss plc

0 1988

Immunoglobulin Dual Genotype Kappa-secreting P&VI

PELTOMiiKI,*

*Department Radiotherapy

analysed

of Medical

and Oncology,

cytogenetics,

Abstract-The

Kappa and Lambda Light Chain Rearrangement in a Patient with B-CLL NESTOR 0. BIANCHI,t SAKARI KNUUTILA,* LASSE TEERENHOVI,: ERKKI ELONEN,§ RITVA LESKINENII and ALBERT DE LA C:HAPELLE*

Genetics,

C:niz)ersity of Helsinki,

University of Helsinki,

immunophenotype

Clones of

K

~LMBICE,

La Plata,

ilrgentina,

:Department

of

of Medicine,

University of Helsinki,

Finland

and /Transplantation.

Laboratory,

University of Helsinki,

Finland

gene rearrangements

or bmphatic

were

leukaemia.

One

of the constant p., K and h genes. The was K-secreting indicating expression of one immunoglobulin light K allele.

immunophenotype

showed monoclonal

with lymphoma

Finland,

$Third Department

and immunoglobulin

in a consecutive series of 56 patients

patient with B-CLL

Finland,

producing

B-cells

interpreted as a failure

rearrangements

usual& show germline

h genes.

The present case may thus be

of the mechanisms controlling isotypic exclusion at the gene level. Further

studies are needed to determine the frequency

of such events and whether the patients show distinct

clinical or otherfeatures.

INTRODUCTION genes

IMM~:N~OL~BIJLIN

arc organized

as discontinuous

that must be brought differentiation (IGH)

joining

several

(C) genes;

constant

DJ

(V) segments

rcarranged

chain

(IGL)

bination sociates nation

of IG genes allele cannot

On the other

gene,

recombination.

In

and

going

both

the other

cases

allele only

is silent

not

allele

light

chain

encoded

cells, the two different isotypes

(K and

by one pair of IGLK alleles

Accrpted 16 February 1988. Corrcspondencr and reprint requests Department of Medical Gem-tics, Helsinki, Finland.

gene.

immunoglobulin

A) arc

known

gene

that

genes

arc

and with A genes

K

allele

However,

there

about

variations

in

information

this

failed

available

[7-91.

and

(B-CLL) having

This

example

hierarchical

which

besides

a productively suggests

of IGLK genes

is the

by the model. WC B-cell chronic lymK-

being

rearranged

allele also had a rearrangement

Chse report The patient 1233

[6]. The

supports

MATERIALS to: Pjivi PrltomSki, M.D.. Haartmaninkatu 3, 00290

under-

cells that

evidence

vent IGLA from undergoing

and by six pairs

follows

of rearrangement

rearrangement

to be

genes

rearrangements

only in those

a productive

leukaemia

secreting IGLK

In human

of IGL K

with

those of A genes,

much

phatic

is

exclusion)

21.

Cl,

shown

IGLK

[5, 61. It has been proposed

scqucnce of events predicted report here one case of human

undergoes

one

(allclic

order

experimental model

the

rearrangement.

second

of

in murine

with IGLA genes in germ-

recombination

recombination

to produce

recombi-

if the first allele fails to produce the

has

of

associated

analysis

genes

malignancies

of IGLK genes

somatic

preceding

recom-

gent,

always

a hierarchical

light

first in one allele and if it

rise to a productive

start or continue

hand,

a functioning expressed

starts

in giving

that

only rcas-

Somatic

B-cell

rearrangements

deletions

with the

Hence,

gene expression

one V with one J segment.

is successful other

IGL

[3, 41. The these

configuration. Conversely, productive rearrangements of IGLA genes are almost always associated with aberrant rearrangements or

5’ of

Immunoglobulin

lack D segments.

preceding

gents

one of many

associated

human

almost line

is fused

located

thereafter,

respectively involving

productive

for gcnr chain

(J) segments

segment. genes

heavy

becomes

and

of segments

(D) segments

alleles,

rearrangements

B-lymphocyte

prerequisite

diversity

to one of several variable

during

F’or immunoglobulin

one of several

configuration

families

together

as a necessary

expression.

of IGLA

in germ-line

of one IGLA that

successful

does not always

pre-

recombination.

AND METHODS

is a 56-year-old

malt

who presented

P. Peltomiiki et al.

1234 in February

1985 with enlarged

neck, supraclavicular lymph

nodes

were

haematologic white

fossae, about

values

blood

nodes

in the

and groin.

2 cm in diameter.

were:

cell count

lymph

axillae

X log/l,

BRL,

Gaithersburg,

fragments gels

Blood

haemoglobin

343.0

The

129 g/l, (differential:

and

capillary

in a mixture

ential

sperm

A biopsy

of a lymph

histology

of a small

diagnosis

of chronic

made.

node

lymphocytic

A

leukaemia

mg daily was started

1985 and the dose reduced

a

lymphoma.

lymphocytic

Chlorambucil6

was

in April

to 2 mg daily in October

1985 the haemoglobin

white blood cell count 93% small lymphocytes approximately

the

condition

followed

were

1985. His essentially

were made

radioactive

the

were above

for lymphocyte

cytogenetic

analysis

occasions

between

The

were

cells

chromosome

used

hybridization

were

at 65°C. h at

for

CD2,

and

Bl

of

analysis. marker

stud-

using

FACS

analysis antibodies

anti-kappa

used

and

to the CK light-chain fragment

Cl.r frag-

gene

[ 121,

homologous

to

detects

deletions

The

were

of pokewced

Surface

marker

chain

clonality

kappa

positive

lambda

analysis in the cells

positive

revealed

blood. was

cultured mitogen

85%

cells was 5%.

Bl and OKT-11

positive

Chromosome successive

Biochemicals,

P-L

or

12-O-tetradecanoylphorbol-13-acetate

(TPA;

2 pg/ml,

any mitogen.

G-banding

for 3 (PWM;

100 p,g/ml,

Milwaukee,

St. Louis,

MO,

Our methods

have been described

revealed

leukaemic blood

mononuclear

tures. or

for fixation

and

[lo].

by one-step

DNA

density

gradient

from

ccntrifug-

ation in Ficoll-Paque (Pharmacia Fine Chemicals, Uppsala, Sweden). To obtain control DNA, fibrocultures

were

initiated

from

a skin biopsy

of

the patient. (5 Fg)

were

digested

overnight

with 30-40 U of the appropriate restriction nuclease. Assay buffers were those suggested (Promega,

Madison,

WI,

U.S.A.;

of

that

of

proportions

performed

In October

of lo%,

that

only cells with in both

PWM-

endoby the Gibco-

and

encountered

2 years

out of 20 mitoses Both

with TPA

abnormality.

a normal

1987 a clonal

46.

on three 1985 a nor-

stimulated

chromosome

In May

were

TPA-stimulated abnormality

earlier

stimulated

One

karyotype

cul-

similar

was found

with TPA.

number

of the abnormal

homologous

chromosomes

clone 11 and

of chromosomes

4, 6 and

abnormal.

karyotypc

be interpreted

The

could

46,XY,t(4q-;llq+),6q-,llq-,12p?The breakpoints involved

to

in four

one homologue

in the

12 were as

(Fig. 1A). t(4;ll) translo-

cation were most likely 4q12 and 1 lq13. Thus, the breakpoints were presumably different from those observed

samples

light

was found in all 35 PWM-stimulated

The chromosome

was extracted

cells separated

whereas The

were

samples.

a clonal

observed

was Southern hybridization analysis.

kappa percentage

cells were 90% and

studies

blood

year later

WI,

U.S.A.),

previously

The

Cytogenetic study

NJ,

Electronics,

U.S.A.)

Sigma,

[ 131.

respectively.

Raritan,

(Coulter

cells

ofCK genes

RESULTS

mal karyotype

in the presence

suppliers

1.3 kb the

were:

anti-lambda

Corporation,

for CD20

Cytogenetic stu&.

DNA

a to

a 2.5 kb EcoRI-EcoRI

cells but two out of 11 mitoses

blast

were:

Surface marker analysis

analysis,

U.K.).

whole

were

intensifying

homologous

a 3.5 kb EcoRI-Hind111

four

or by immunopcroxidase

monoclonal

Pharmaceutical

U.S.A.),

from

using

cmploycd

gene [Ill,

homologous

SDS, respectively.

Autoradiograms

-70°C

fragment

heavy-chain

SDS,

of 30 min each in 1 x SSC,

probes

EcoRI-EcoRI

at

Filters

1987.

extraction

Immunologic

Dickinson)

The

OKT-11

and

by fluorescence

(Becton

on May

marker

analysis

Surface marker analysis.

staining.

obtained

1985 and

for surface

banding

ies were done

without

by nick

performed

characterization

wcrc

October

DNA for Southern

days

herring

solution.

SDS and 0.1 X SSC, 0.1% washes

ment

solution,

denatured

for 30 min with 2 X SSC, 0.1%

(Kde). This probe samples

(Ortho

mem-

10 X Denhardt’s

by two washes

The

nodes

to nitrocellulose

the Chl_6 light-chain genes [4], and a 2.5 kb BamHI-Hind111 fragment designated K-deleting

Blood

Luton,

were washed

screens.

lymph

DNA agarose

50 kg/ml

h in

343.0 X log/l, (differential: and 7% granulocytes) and

Methods

IV

20-24

for 24-72

The

Digested or 0.6%

hybridizations

exposed

size as in February since remained

has

Probes and

for

0.1%

unchanged.

and

65°C

and

143 g/l,

119 X log/l.

was

DNA.

translation

All

1985. In October

platelets

in the neck revealed

blotted

X SSC,

of4

0.1 M NaH2P04

70% small lymphocytes,

U.S.A.). in 0.8%

branes (Schieicher & Schuell, Dassel, F.R.G.). Prehybridizations were carried out overnight at 65°C

97% small lymphocytes and 3% granulocytes) and platelet count 191 X log/l. A bone marrow differshowed

MD,

were separated

in the B-cell/myeloid

mixed

leukaemia-

specific t(4; 1 l)(q21;q23) translocation. However, a breakpoint in 1 lq13 is seen in t(l1;14)(q13;q32) occurring in B-CLL. The 6q- and llqchromosomes

were

mctacentric

and resulted

from translo-

1235

IGLK +h Bigenogpe

A

B 22

14 1;i.q. 1, Portia/

(;-bonded

kay@pe.

Chromoromec genes (H).

inooll,ed in the rlonal abnormali&

.-Irrowtzndicate

12 -

(A) and those contniniq

the abnormal homo1ogur.r.

-

A 12

/he rmmurqlobulm

P. Peltomiiki et al.

1 -

2 -

-

-

-

IGLK+~ cations

with

rather being

at

and

undetermined

than

deletions,

6ql3

and

22 containing

carefully observed than

other

the llq13.

rearrangement

chromosomes

breakpoints

probably

Chromosomes

the immunoglobulin

2,

genes

ment 14

were

set (Fig.

1B).

germ-line DNA the

digestion

and

fragment

of

sample B-CLL

BamHI

lane

DNA

bands

1). The

the Kdc probe

with sample

digestion

lane

of one

and

the leukaemic

2.5 kb germ-line rearranged

and

band

DNA from normal The Ch probe

white used

fragments

14, 8 and

at

16,

cross-hybridization

cells. detects

germ-line

The

bands

5 kb

polymorphism

is known fragment

sample

a novel

showed

fragments

(Fig. 4, lane

rearrangement

of the EcoRI

on

three

exhibit

suggests

leukaemic

DNA

suggests

20 kb allele.

all the

at

6 month

intervals.

sidered

DISCUSSION CLL

consecutive

patient series

malignancies

by the clone,

findings

studied.

of monoclonal of one

the expression

belonged with

Circulating origin

chain

B-cells

with

bacterial

been

from of clones

chains

exhibiting

and

marker analysis, and the presence of one rearrangement by Southern hybridization.

clonal This

suggests that rearrangement

IGLK IGLA

B lymphocytes gent

is con-

appearance during and responsiveness [ 191. Lymphoma

B cells

of these

and

stimulated

produce both

of IGLK

cells express

although a failure

rearrangements.

a low

K and A light

It

and

only A

K message

and

Some

Ly-1

in the cytoplasm.

may exhibit

deter-

characteristic

rearrangement

on the surface

of IGL

in germ-

B) that

with

expressing

light chains

abnormal

(Ly-1

described Ly-1

malig-

can be found

A gene,

A genes

lineage

[20]. Most

protein

revealed

of one

lipopolysaccharide

proportion

IGL allele.

has not so far been

IGLA genes

by surface

the coincidence of a productive plus a non-productive

has

derived

rearrangements ofone

their

surface antigen expression, devclopmcnt, tissue distribution cells

be

frequency of K and A dual in K-secreting human lym-

and

as cvidenccd

chromosomally

of one light

to a

lymphatic

the immunophcnotypc

were

cells were

here

of 56 patients

in whom

immunogenotypc nant

described

gene

plasmacytomas

had

of normal

it may

of the mcchan-

rearrangement others

dual

Since the

et al. [5], one cell line out

murinc

to be a specific

to antigens

The

IGL

and leukaemias

X subset were idcnt-

exhibits

of failure

by Coleclough

of 14 K-producing

phomas mined.

[17, 181. by which

the rearrangc-

here

rearrangement

line configuration. The genotype rearrangement a

genes

kappa-secreting,

further

a non-productive

no 20 kb

pattern

blocking

was

after the successful

while

non-B,

of B-CLL ofimmuno-

(TCR)

reported

as an example

in

10%

of IGLK and IGLA genes.

clone

1).

and

genes or vice versa.

patient

In a study

somatic

of the mechanisms

considered

an EcoRI

VJ

they arc not

of an IG gene blocks

of TCR

isms

gene

dual rearrangements

a failure

rearrangements

at

genes

regulatory rcarrangc-

per cent of non-T,

and T-ccl1 receptor

a rearrangement

contained

occasions

Thirty

IG

However,

leukaemias

malignant

from

above

controlling

from

4 kb but

described

subsequent

error-free. lymphatic

ment

bc seen

of IGL

of a functional

are effective.

with

(5 kb [4, 141.

of a

recombi-

[ 15, 161. We assume

exclusion

occurrence

mechanisms

with

of 9.6 kb, germ-line

2). This

The rearrangements

The

lcukacmias

(Fig. 4, lane

[4]. The band

allele

CA,_,; genes

also

to result

of 16, 14, 8, 5 and

fragment

ical

could

the

recombination

This

a pscudogenc localization

of one of the CX gcncs

the 8 kb EcoRI

after

CK

resulting

IGH isotypic

of a con-

assembly.

acute

germ-lint

with

and

the existence

be mediated by a negative that prevents further IGL

The

20 kb in the fibroblasts

20 kb band

in the other

deleted

EcoRI

of the probe

(16, 14 and 8 kb bands) and band) of unknown chromosome

inhibits

globulin

observed

indicating

cxper-

the appearance

allelic

and

direct

by which

the

was likely due to contamination

is now

rcarrangemcnt

suggest

one

There

V,DJ,

totally

exhibited

a rearranged

data

The very weak germ-line

the CK probe

hybridization

plus

These

one

(Fig. 3A,

DNA

bands

2).

showed

allele

gene.

assemble

mechanism

functional

mcnts

(Fig. 3A,

to

a given B-cell clone IGH gene and one

trolling

may also mechanism

the CK probe

potential

all IG genes. Yet, only one functional IGL

B-cell

transformation.

evidence

that

rearranged

the

rearrange-

rearranged

imental

nation

1) while

of 12 kb

DNA

BamHI

(Fig. 3B,

4 kb and

a

fibroblast

two

and one rearranged

2). After

Additional

the

showed

fragment

lcukaemic

lane

allele.

in

(Fig. 2, lane

hybridized

with

presence

produced

(Fig. 2, lane 2).

a BamHI

14 and

19.6 kb

sample

DNA

weak germ-line

band

probing

of the patient

Fibroblast exhibited

Cp

malignant

have

express products

of a second

in a productively

that underwent

functional

IG gene rearrangemelds BamHI

was the result

occurring

B-Cells

studied. However, no abnormalities were in those chromosomes at the level of less

300 bands/haploid

1237

Bigeno(@e

of the control remains

to be

determined whcthcr the patient we report rcprcscnts malignant transformationofI,y-1 B cells. Moreover, further studies are nccdcd to find out whether these malignancies

represent

a specific

clinical

subtype.

I238

P. Peltomiiki et al.

Acknowledgements-\e thank Dr. Philip Leder, Department ofGenetics, Harvard Medical School, Boston, MA, for providing us with the QL, CK and CA probes. We also thank Dr. Stanley J. Korsmeyer, Department of hledicinc, Microbiology and Immunology, Howard Hughes Medical Institute, Saint Louis, IMO,

for providing the Kde probe. This study was supported by grants from the Sigrid Juselius Foundation, the Academy of Finland, the Folkhalsan Institute of Genetics, and the Finnish Cancer Society.

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11.

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K

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Proc Nat1 Acad Sci USA 1986, 1986, 1438-1442.