Download Hairy Cell Leukemia Is Characterized by Clonal Chromosome

From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
Hairy Cell Leukemia Is Characterized by Clonal Chromosome Abnormalities
Clustered to Specific Regions
By Ulla Haglund, Gunnar Juliusson, Birgitta Stellan, and Gosta Gahrton
Cytogenetic analysiswas performed on B-cell mitogen-stimulated cells from 36 patients with symptomatic hairy cell
leukemia. Evaluable metaphaseswere achievedfrom 30 pa(67%)
showed clonal abnormalities. Recurrent
tients, and 20
chromosomal aberrations involving chromosomes1,2,5,6,
11, 19, and 20 were found. The abnormalities were mostly
deletions andinversions,whereastranslocationsandnumerical abnormalities, except
trisomy 5,were rare. Fourteen
patients showed multiple clones, which mostly were unrelated and found in different combinations in individual cells.
Cells with non-clonal abnormalities identical
to those found
in clonal changesin other patients were common. Chromosome 5 was involved in clonal aberrationsin 12 of 30 (40%)
patients, most commonlyas trisomy 5 (n = 4). or pericentric
inversions (n = 6)and interstitial deletions (n = 4) involving
band 5q13.Three patients showed two and 1 patient three
different clones that involved chromosome5. In addition. 1
patient had a rare constitutional inversion of chromosome
5 with breakpoints at p13.1 and q13.3.Pericentric inversions
and interstitial deletions of chromosome
2 occurred clonally
in 4 patients (13%) and in single cells of another6 patients.
Deletionsofchromosome 1 at band q42 was found in 5
patients, and 1 patient had a translocation between lq42
and a supernumerary chromosome 5. Deletions of 6q and
1 Iq were similar to those commonly found
in other lymphoproliferative disorders. Trisomy 5, structural abnormalities
5
involving the pericentromericregionsofchromosomes
and 2, and lq42 abnormalities were findings distinguishing
the karyotypes in hairy cell leukemia from those of other
hematologic malignancies.
0 1994 by The American Society of Hematology.
H
previously published.' Cells from 18 patients were sampled before
treatment, while 13 had been splenectomized and/or had received
prior treatment with interferon a (IFNa).Five patients had received
multiple regimens, including 2-deoxycoformycin or chlorambucil,
before sampling. All patients had active disease, ie, any type and
degree of cytopenia with or without symptoms and infectious complications, and all patients were subsequently treated with 2-chlorodeoxyadenosine.'.''
Cell cultures and mitogens. Cells from peripheral blood and BM
were cultured in RPM1 1640 supplemented with 10%fetal calf serum
(GIBCO, Paisley, Scotland) or 10% human pooled AB serum from
regular blood donors, at a cell density of 1 to 2 X 106/mL.In
two patients, fibroblasts from skin biopsies were cultured because a
constitutional abnormality was suspected.
B-cell mitogen-stimulation wasin most cases performed using
BSF-MP6, the supematant from a human T-T hybridoma cell line
(a generous gift of G. Kjellstrom and A. Rosin, Karolinska Institute,
Stockholm, Sweden). MP6 was used at 25% (voYvol) in medium
with added heat-killed, formalin-fixed Staphylococcus aureus Cowan
strain I (SAC) (1:80 OOO, generously donated by M. Carlsson, Pharmacia AB, Uppsala, Sweden), and human recombinant IL-2 (rIL-2)
(10 U/mL, Genzyme, Cambridge, MA).I4 In addition, combinations
of lipopolysaccharide (LPS) (100 pg/mL, Sigma, St Louis, MO) and
4P-phorbol 12P-myristate 13a-acetate ( P A ) (2.0 pmol/L, Sigma),
or LPS and cytochalasin B (CB) (1.O pg/mL, Aldrich Chemie GmbH,
Steinheim, Germany) were used.I5 Pokeweed mitogen (PWM) (5.0
pg/mL, GIBCO, Grand Island, NY) and the supernatant from the
Epstein Barr virus-containing B95-8 cell line were occasionally used.
AIRY CELL LEUKEMIA (HCL) is a chronic lymphoproliferative disease' constituting 2%of the leukemias. It affects mostly males, with a yearly incidence of six
per million. The disease is clinically characterized by anemia, granulocytopenia, thrombocytopenia, monocytopenia,
splenomegaly, and impaired immunity. The course might
thus be fatal because of an increased risk of opportunistic
infe~tions.2'~
Bone marrow (BM) fibrosis is common, and
BM morphology is diagnostic. Hairy cells are large differentiated B cells, often exhibiting hair-like protrusions. The
hairy cell phenotype is peculiar, with a common expression
of the B-cell markers CD19 and CD20, multiple Ig heavy
chains: HLA-DR, CD45RA, the monocyte marker CD1 IC,
and the interleukin-2 (IL-2) receptor CD25, but occasionally
also of other markers, such as CD4, CD5, CD15, or CDlO.'
Regarding the karyotype, few investigations on chromosome abnormalities in HCL have been reported. This might
in part be explained by the rarity of the disease and the
difficulty in obtaining cell material caused by the usually
low number of circulating hairy cells and the BM fibrosis.
Initially unstimulated cells were studied:.'
and a few numerical abnormalities, including trisomy 12, were found. When
B-cell mitogens were introduced, more clonal aberrations
were observed, most commonly structural abnormalities involving chromosome 14, such as 14q deletions' and 14q+
marker chromosomes." However, no consistent abnormality
was linked to HCL. Furthermore, the overall success rate
was low, with clonal abnormalities detected in 38 of 153
(25%) studied patients.'' Thus, we were prompted to perform
the present investigation, in which several recurrent abnormalities were found. Chromosome abnormalities in regions
commonly involved in other hematologic tumors were
found, but also those distinguishing HCL from related disorders.
PATIENTS AND METHODS
Patients. Cytogenetic analyses were performed on cells from 36
patients, 34 men and 2 women, with a mean age of 55 years. The
diagnosis was based on typical findings in BMbiopsies and aspirates
with cytochemistry, supplemented with flow cytometry using a large
panel of antib~dies.~.~.'*
Clinical data from 16 of the patients were
Blood, Vol 83, No 9 (May l), 1994 pp 2637-2645
From the Division of Clinical Hematology and Oncology, Department of Medicine, Karolinska Institute at Huddinge Hospital, S-l41
86 Huddinge, Sweden.
Submitted October 5, 1993; accepted January 3, 1994.
Supported by the Swedish Cancer Society, The Swedish Society
for Medical Science, The Karolinska Institute's Research Funds,
and The Medical Research Council.
Address reprint requests to Gunnar Juliusson, MD, Department
of Medicine, Huddinge Hospital, S-l41 86 Huddinge, Sweden.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1994 by The American Society of Hematology.
0006-4971/94/8309-0034$3.00/0
2637
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
HAGLUND ET AL
2638
Table 1. Patients With Constitutional, Clonal, and Nonclonal Recurrent Chromosome Abnormalities
Patient Age1
Previous
Treatment*
AbnormalWotal
Cells With Constitutional, Clonal, and Nonclonal Recurrent Abnormalities
Cell Source1
Mitogenst
No. of Metaphases:
No.
B/
9.90 TO§
49 yrs/S, I, C
PWM
karyotype:
Constitutional
211 1
Patients with clonal abnormalities involving chromosome 5
17.92 IK
38 yrs/U
BM, B/
MP6
3.91 RH§
39 yrs/S, I
7.91 GE
50 yrs/S, l
BM. B/
LPS, TPA, CB
BM/
MP6, LPS
TPA, CB
6.92GS
64 yrs/U
BM/
MP6
1.90 LHS
51 y r d l
2.92 KK
44 yrs/U
B/
LPS, TPA, CB
BM. B/
MP6
14/37
5
2
1
2
2
1
416
2
25/47
1
16
1
1
1
3
1.91GP§
73 yrs/S, l, P,
5.91DK
36 yrsll, dCF
7.90 HEJS
43 yrsll
C
23/25
3113
17/43
BM. B, LW
LPS, CB,
MP6, TPA,
PWM
14/36
BM, B/
LPS, CB
TPA
2 1/89
BM. B/
LPS. CB
"PA
15/29
8
8
1
2
1
2
1
1
1
1
1
1
1
1
2
1
2
3
1
1
1
1
1
1
1
2
1
1
1
1
2
2
1
2
1
l
1
2
2
1
fcontinued on following page)
Karyolype
46.XY,inv(5)(p13.lq13.3)~
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
2639
CHROMOSOMEABNORMALITIES IN HCL
Table 1. p a t i e m With Constitutional, Clonal, and Nonclonal Recurrent Chromosome Abnormalities (Cont'd)
Cells
Age1 Patient
Source/
Cell
Previous
Treatment*
genst
Abnormalities
Recurrent
Nonclonal andWithClonal,
Constitutional,
No. of Metaphases:
KaryoWpe
No.
Patients with clonal abnormalities involving chromosome 5 (cont'd)
19.87 KS
74 yrs/U
35.92
LS
69 yrs/U
6.90 JJS
56
yrs/S,
I, dCF
B/
LPS, CB
TPA
BM, B/
MP6
BM, B/
LPS,
TPA
CB, MP6
7114
18/34
3
1
2
4
2
2
20123
1
1
17
1
1
1
Patients with other clonal abnormalities
LT
27.92 JEK
52 yrs/U
BM, B/
MP6
7.92 AW
64 yrJU
BM/
MP6
4.92 SGC
61 yrs/U
BM, B/
MP6
LPS, TPA
10.91RB
46 yrs/l
BM/
MP6
8/36
8/27
9/25
13/20
9.91 SK
49 yrsll
4.91 NM
40 yrs/l
BM/
MP6
BM, B/
TPA LPS,
11/26
12.90 KFJ
73 yrs/U
11.91
58 yrs/U
BM/
CB
TPA,
LPS,
BM, B/
MP6
3111
9/33
7118
2
de1(2)(qllq13)or(q13q21)
2
de1(2)(qllq13)or(q13q2l),add(ll)(p?)
1
del(2)(q?q?)11,de1(6)(q25)11
1
inv(2)(pl1-13q11-13)"
constitutional karyotype: 45,XY,t(l3;14)(q10;q10)c
2
1
1
1
1
1
1
1
1
1
1
1
2
1
1
2
1
2
1
3
1
1
1
2
2
1
Patients with nonclonal recurrent abnormalities only
BA
214
6.91
49 yrdl
21.92 IM B/
48 vrs/U
3114
CB TPA,
LPS,
BM,
MP6
1
1
1
1
.
.
Previous treatment: U, untreated; S, splenectomy; I, interferon; dCF, 2'-deoxycoformycin; C, chlorambucil; P, prednisolone.
t Evaluated cell sources and cultures: BM, bone marrow; B, blood; LN, lymph node. LPS,CB,TPA,MP6,
PWM are mitogens, see text. The
bold font indicates cultures in which clonal abnormalities were identified. Unmarked cultures include those with no metaphases.
The number of abnormal metaphases includes clonal, nonclonal recurrent, and sporadic aberrations, but not constitutionalabnormalities.
§ Patients previously reported in ref 3.
Nonclonal aberrations that occur as clonal aberrations in other patients.
*
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
2640
HAGLUND ET AL
To obtain elongated chromosomes of normal T cells from patient
9.90, ethidium bromide (10 pg/mL) during the final 2 hours of a
phythemagglutinin (PHA)-stimulated lymphocyte culture was
used.“
The cells were harvested according to medium color change over
a number of days. In general, cells were harvested on days 4 a n d
or 5 , with additional harvests in some patients after up to 25 days
in culture. Colchicine (Demecolcine, Sigma) to a final concentration
of 1.25 or 0.125 p m o m was added 1 hour before harvest. The
cells were treated with a hypotonic (75 mmol/L) potassium chloride
solution, and fixed in acetic acidmethanol. Slides were, in general,
flame dried to improve the spreading of the metaphases.
Cytogenetic analysis. Conventional Q- and G-banding techniques were used.”,18 Cytogenetic analysis was performed with a
Cytoscan Image analysis system (Applied Imaging, Sunderland,
UK). Chromosome abnormalities were given, and clones were defined according to the International System for Human Cytogenetic
N o m e n ~ l a t u r e .Two
’ ~ ~ ~cells
~ with identical structural abnormalities,
two with identical supernumerary chromosomes, or three with the
same missing chromosomes constituted a clone. Single cell abnormalities that appeared as clonal changes in other patients were referred toas “nonclonal recurrent” aberrations. We also regarded
nonclonal abnormalities involving recurrent regions, but
with
breakpoints that did not exactly coincide with those of clonal aberra-
tions, as in the case of the pencentric inversions of chromosome 2,
as “nonclonal recurrent.”
RESULTS
In samples from 6 of the 36 patients (17%) no metaphase
was found. Twenty male patients of 30 evaluable (67%)
showed clonal abnormalities and another two had nonclonal
recurrent aberrations (Table 1). Eight patients showed no
clonal abnormalities, butonly sporadic chromosome and
chromatid aberrations. No evaluable patient lacked chromosomal abnormalities entirely. One patient had a constitutional abnormality that was also found as a clonal change in
other patients (see below). Most patients exhibited a large
number of different chromosomal abnormalities. The abnormalities were mainly structural, and inversions and deletions
werenotably more commonthan translocations. Unstable
aberrations, such as dicentrics, rings, and chromatid aberrations also appeared. There were few numerical abnormalities.
The chromosomes preferentially involved in clonal abnormalities were Nos. 1, 2, 5, 6, 11, 19, and 20 (Fig 1). Clonal
changes involving chromosomes 4, 12, 13, 14, and 15 were
1
2
6
5
bb
%-U
I
I
11
47 ++++++++
19
20
Fig 1. The locationof clonal and nonclonal recurrent
abnormaliiiesin the most frequently affected chromosomes.*A sporadic chromosome
aberration includingat least a part of a region involvedin clonal abnormalities, althoughnot with exactly coincident breakpoints. **Constitutional inversion of chromosome 5.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
CHROMOSOME ABNORMALITIES IN HCL
2641
Fig 2. Typical chromosome abnormalities. The
abnormal homolog is to the right and the affected
regions indicated by arrows on the normal homolog
to the left. la) inv(5)(p13.lq13.3)as a constitutional
abnormality in patient T6, (b) +inv(5)(pl3ql3)as an
acquired abnormality, (c) del(5)(qllql3)or(ql3ql5),
(dl del~2l(qllq13~or(q13q21),
(e) deI(ll)(q13q21), (f)
inv(19)(p13q13).
occasionally found. No correlation between the number of
clonal abnormalities and the number of metaphases investigated was observed. Cells with clonal abnormalities were
frequently seen in multiple cultures, if available (Table l).
In our study, MP6 was the single most effective mitogen for
hairy cells. No significant impact of previous treatment on
the chromosome findings was found, but three of five patients with multiple previous treatment regimens had clonal
abnormalities involving chromosome 5 (Table l).
Chromosome 5. Thirteen of the 30 patients (43%) with
evaluable metaphases showed clonal (n = 12) andor nonclonal recurrent (n = l ) abnormalities of chromosome 5
(Table l). One patient showed three, and three patients two
different clones involving chromosome 5. The most commonly occumng abnormalities were inversions (n = 7 including the constitutional inversion, see below) (Fig 2a and
b) and interstitial deletions (n = 4) (Fig 2c), all except one
involving band 5q13. Supernumerary normal (n = 4) and
derivative (n = 4) chromosomes 5 were also common. An
additional patient with a del(5)(ql lq13) clone had single
cells with trisomy 5 and t(5;13)(q13;q?), respectively,
among 89 studied cells. One patient with trisomy 5 had two
additional clones with extra chromosome 5 derivatives from
a t(1;5) (q42;p15.1) and an inv(5)(p13q13), respectively.
Five of the six pericentric inversions included band 5q13
and three of them were found in supernumerary chromosomes (Fig 2b).
Sporadic aberrations also involved the 5q13 region: one
terminal deletion in 5q13, the t(5; 13)(q13;q?) mentioned
above, and one insertion of the segment 5(qlI-l3q22) into
a chromosome 8. On the other hand, sporadic translocations
involved bands pl, p15, and q35, similar to the sites of clonal
translocations (see below).
One additional patient had a constitutional pericentric inversion, with the breakpoints p13.1 andq13.3, as defined
by the use of high-resolution banding.'" The inversion was
present in all metaphases studied from blood and BM during
active disease and in complete remission, as well as from
skin fibroblasts, whereas no other clonal aberration was seen
in the eleven metaphases achieved from BM when involved
with HCL.
Clonal translocations (n = 3) affected the telomeric parts
of either arm, with breakpoints in 5 ~ 1 55q31,
,
and 5q33-35.
Chromosome 2. Clonal andor nonclonal recurrent interstitial deletions (Fig 2d) and inversions involving the pericentric region of chromosome 2 were found in 10 patients
(33%). The region common to all these abnormalities was
band 2q I 1 (Fig l ) . Because the banding pattern in this region
is not very distinct, the exact location of the breakpoints is
somewhat uncertain. The true incidence of these aberrations
may be higher because of a conservative assessment of the
abnormalities.
Chromosome I . Terminal deletions of chromosome 1 at
band q42 were found as clonal and nonclonal aberrations in
three and two patients, respectively. A translocation between
lq42 andband p 15.1 of a supernumerary chromosome 5
(see above) was also found.
Chromosome 6. Clonal terminal deletions involving
band 6q25 occurred in four patients, and as non-clonal abnormalities in another five. One patient exhibited deletions of
the long arm with different break points in different cells:
q21-23 in one metaphase, q23-25 in two, and q25 in one
cell. This patient also had a clonal pericentric inversion comprising most of the chromosome, with breakpoints at p25
and q25.
Chromosome 11. Chromosome I 1 was, together with
chromosome 5, most frequently involved in clonal abnormalities, such as terminal deletions in band I lq23 and interstitial
deletions with the breakpoints q 13q21 or q21q23 (Fig 2e).
Pericentric inversions, one clonal and one nonclonal recurrent, involving the break points pl3- 15 and 92 1-23, occurred
in two patients. In addition, eight patients showed additional
material, the origin of which could not be identified, on the
short arm.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
HAGLUND ET AL
2642
Chromosome 19. Clonal pericentric inversions were
found in cells from three patients, and in a further two as
nonclonal aberrations (Fig 20. One patient exhibited a ring
chromosome with undefined breakpoints.
Chromosome 20. Clonal terminal deletions of the long
arm of chromosome 20, all including the region q13.1 to
qter, were found in two patients, and as nonclonal recurrent
changes in another two.
Numerical abnormalities. Only a few numerical abnormalities were present, except for theadditional chromosomes
S (see above). Two patients showed an additional
del(l2)(pl1) derivative
chromosome,
and one patient
showed clonally missing chromosomes 7 and 21 together
with a marker chromosome.
Clonalbreak points. Clonal break points, ie, identical
breakpoints involved in different aberrations, clonal or nonclonal, in the same patient, were identified in ten patients.
Band Sq13 was involved in different abnormalities in two
patients. Also chromosomes 11 and 19 showed terminal and
interstitial deletions, inversions, and translocations with coincident break points. One patient showed a deletion in 6q2S
and a chromatid break in the same band.
Multiple clones. The frequency of cells with clonal abnormalities varied greatly, from 20 of 23 cells (87%) in one
patient to zero in others. Fifteen patients had more than one
clonal aberration (Fig 3) and 14 of these exhibited distinct
multiple clones. Multiple clonal abnormalities often coexisted in different combinations in different metaphases. This
is exemplified by one patient with 17 aberrant metaphases
of 43 studied; 10 of these contained six different clonal
abnormalities in various combinations with other abnormalities, so that no two metaphases had identical karyotype.
Constitutional abnormalities. Two patients were found
to have constitutional abnormalities, both documented
through karyotyping of skin fibroblasts. One patient had an
inv(S)(p13.lq13.3), without other clonal changes (see
above). The second patient had a Robertsonian translocation
between chromosomes 13 and 14, and additionally a clonal
pericentric inversion of chromosome 2 in leukemic cells.
I
0
1
2
3
4
5
6
Number of abnormalities
Fig 3. The number of patients with various numbers of clonal and
nonclonal recurrent abnormalities.
DISCUSSION
Our investigation presented a high frequency of clonal
abnormalities in HCL. These were in part abnormalities that
are common in different hematologic tumors, and previously
described in HCL. However, some of the most frequent abnormalities in our study are rare in other types of leukemias.
In contrast to the case in most other hematologic tumors,
the structural abnormalities were mostly caused by inversions and deletions, but rarely translocations. Multiple clones
and different types of abnormalities involving the same chromosome region in individual patients were other characteristic features.
In previously published investigations comprising more
than 150 HCL patients, one fourth of the patients that were
evaluated, had clonal abnormalities," as compared with two
thirds in our study. Recently, fluorescence in situ hybridization techniques, permitting sensitive detection of numerical
chromosome abnormalities in interphase cells,have been
applied to 24 cases of HCL?' without significant findings.
However, no probe for the detection of chromosome 5abnormalities was used. In our study, abnormalities involving
chromosomes l , 2, and 5 were the most significant findings.
These were typically trisomy 5, structural abnormalities involving band 5q13, pericentric inversions and deletions of
chromosome 2, and structural abnormalities of band lq42.
Structural abnormalities of chromosome 5 are previously
reported in three HCL cases; in two of them band p1 was
involved.'"~zzA clonal 5q13.3 breakpoint is reported in one
patient with a variant form of HCL as part of a three-way
translocation, t(1;S; lO)(p34.3;q13.3;q22.1), with an additional t(S; 14)(q31.1; q l l ) sub-clone." Trisomy 5, pericentric
inversions of chromosome S , and other structural abnormalities involving 5q13 are rare in malignant lymphoma and in
chronic lymphocytic leukemia (CLL)," the malignancy most
closely related to HCL. Of S12 CLL patients with clonal
abnormalities included in the database of the International
Working Party on Chromosomes in CLL,",24 14 (2.7%) involved chromosome S , mostly as t(1;S) translocations. No
single CLL patient had 5p13 abnormalities or pericentric
inversions of chromosome S . Trisomy 5 was found in conjunction with other trisomies in 1 CLL patient, and clonal
breaks in band 5q13 were found in 3 (0.6%) (G. Juliusson,
personal observation, 1993). In similar, int de1(2),
inv(2)(pl lq13), and del(l)(q42) are reported in single cases
of CLL only." Deletions of Sq, on the other hand, are characteristic features of the myelodysplastic syndromes and secondary myeloid leukemias. However, in these tumors the
abnormalities always involve the distal part of the long arm,
from q22 to q33," a region encoding several cytokines and
cytokine receptors.26
Among numerical abnormalities in HCL, trisomy 3 and
21, and monosomy 7, 10, 12, and 17 have been
In our study, trisomy S was the only recurrent numerical
abnormality; in addition, one patient showed monosomy 7
and 21.
Trisomy 12 is the most common abnormality in
CLL,'','5,".27whereas structural abnormalities involving this
chromosome are less freq~ent.'~
In HCL, trisomy 12 was the
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
CHROMOSOME ABNORMALITIES IN HCL
2643
necessary for tumor formation,@'
exemplified by the consisfirst identified chromosome abnormality,6-' but thereafter it
tent, progressive accumulation of certain abnormalities in
has been found in single patients
Structural abnorcolon carcinoma: a RAS gene mutation, del(5q), del(l7p),
malities on the short arm of chromosome 12,withbreak
and del( 18q):' The recuning chromosomal abnormalities
points in 12pl3 and p1 1 were reported by Brito-Babapulle
found in this study of HCL may enable the identification of
et
and +del( 12)(pl1) was also found twice in our study.
some important gene loci.
Of specific interest was that both these patients with partial
The 5q13.3 band is likely to harbor candidate genes intrisomy 12 also had chromosome 5 and 14q abnormalities
volved in some transforming event of HCL. This is suggest(patients GP and JJ3), and their HCL showed CLL-like feaed by the frequent occurrence of structural abnormalities
tures as regards the phenotype with lack of CD25 expression
involving this region,and the rare constitutional inv(5)
andor CD5-positivity, high blood lymphoid cell counts, and
(p13.lq13.3) abnormality found in our patient TO. The incipoor response. to IFN and deoxyc~formycin.~
dence of constitutional abnormalities is reported to be about
Previous studies have indicated recurrent 14q32 transloca0.6% among almost twelve thousand newbornbabies:' Most
tions and terminal deletions of chromosome 14 with
aberrations were numerical, and balanced structural abnorbreakpoints in the central part of the long arm?*10*22 We
malities affecting autosomal chromosomes were found in
found two patients with clonal chromosome 14 abnormalities
0.2%;' most commonly as Robertsonian translocations in(above). Such abnormalities are found in all B-cell malignanvolving two of the chromosomes 13, 14, 21, and 22, such
cies, and are likely to be unspecific changes in HCL as well
as in our patient AW. Constitutional inversions have been
as inCLL." Deletions of the long arms of chromosome
found in 0.02% of67,000 new born^;^ and in 0.03%of more
6,10*22 and
chromosome 1l''*'' in HCL are also characteristic
than 3,000 adult patients with hematologic diseases.44None
findings in CLL"*" and lymphoma^.^^ Deletions at llq14
of these reported inversions involved chromosome 5, and
and of 20q occur in myeloid disorder^.'^ 19q13, a recurrent
thus the constitutional inversion of our patient TO is exbreakpoint, is the site of the bcl-3 oncogene?' which was
tremely rare.
cloned from a rare recurrent
but
translocation
t(14; 19)(q32;q13) in CLL.3' A few other previously reported The RASA gene45 and the dihydrofolate reductase gene
(DHFR),& both located to 5q13, might be involved in the
clonal abnormalities" were found in single cells from our
process leading to chromosome instability andneoplasia.
patients, ie, de1(2)(q33-35), de1(9)(q22), and del( 18)(q21).
RASA encodes the GAP protein, a GTPase activating protein
The nonclonal recurrent abnormalities in our material might
that binds to RAS p21 proteins, and is implicated as a receswell indicate clones that were not detected because of a
sive oncogene:' Mutant RAS genes may also induce struclimited number of metaphases analyzed.
tural chromosome instability.& The RAS genes are involved
A characteristic feature of HCL cells was the presence of
in the control of cell proliferation, and can be activated in
distinct multiple clones with mostly unrelated abnormalities
most types of human tumors at varying frequencies:' More
that occurred in different combinations in different cells (Tathan 30% of human tumors contain a mutation in one of
ble l), without evidence for a karyotypic evolution. In our
the RAS genes, N-rus, H-rus, and K - ~ u s , ~located
'
at lp13,
material, nearly half (14 of 30) of the patients showed unrellp15.5, and 12~12.1,respectively. Another RAS gene,
lated and partially related clones. Cytogenetically unrelated
clones have been reported in a number of hematologic neoRAB4, is located at 1q42-44." Seventeen of our 30 patients
(57%) showed abnormalities, clonal or nonclonal, in lq42,
p l a s m ~ . ~They
~ - ~ 'have been estimated to be most common
5q13, the l l p , or 12p regions. Therefore, studies of RAS or
in chronic lymphoproliferative disorder^,'^ ie, found in approximately 7% of the cases. The theoryput forward by
RASA genes in HCL would be justified.
Heim and M i t e l m a ~that
~ ~submicroscopic
~
abnormalities link
An inhibition of DHFR,also located to 5q13, could inhibit
the different clones together could explain some, but probatheproduction of thymidine monophosphate, and the rebly not all, of the clonal patterns seen in our patients.
sulting imbalance of the nucleotide poolin the cell may
Specific chromosome regions were also often involved in
cause an increase of chromosome abnormalities and aneumore than one kind of aberration. As an example, a segment
ploidy:' and also affect the induction of folate-deficient fragof chromosome 1, ie, q12q21, was involved in three different
ile sites.52Folate-induced fragile sites coinciding with recuraberrations in the same patient, namely inserted at another
rent HCL breakpoints are located at 2q11.2 andq13,l lq13.3
location in chromosome 1 in one metaphase, inserted into
and q23.3, and 1 9 ~ 1 3 . Twenty-four
'~
of our patients (80%)
chromosome 2 at the critical region ql l-l3 in another metahad clonal or nonclonal abnormalities involving at least one
phase, and interstitially deleted in a third metaphase.''Jumpof these sites or 5q13.
ing translocations" have been described in a few cases of
In conclusion, our study has identified certain chromoleukemias and lymphomas, mainly of the B-cell t ~ p e . ~ ~ -some
~ * regions not randomly associated with HCL. Some of
The probability of an identical translocation, ie, an abnormalthem seem common to avariety of hematologic tumors,
ity with two breakpoints, to occur independently in two difwhereas others, involving bands lq42,2qll, and 5q13, seem
ferent cells has been estimated to be 5 X lo-" at the resolucharacteristic for HCL. Molecular studies of genes localized
tion of the banding pattern used in the present inve~tigation.~~to these regions and their corresponding proteins might enThese data may point to the presence of a common genetiable the identification of tumorigenic mechanisms. HCL is
cally unstable progenitor cell.
probably the systemic malignancy with the best treatment
Tumorigenesis is considered to be a multistep process. It
options, and it would be worthwhile to elucidate genetic
has been estimated thatfive to six independent steps are
mechanisms that distinguish HCL from other tumors.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
2644
HAGLUND ET AL
ACKNOWLEDGMENT
We are grateful to Professor Lore &h, Academic Hospital, Uppsala, for critical reading of the manuscript, and to Dr Anders RosCn,
Linkoping University Hospital, for providing the MP6 mitogen.
REFERENCES
1. Bouroncle BA, Wiseman BK, Doan CA: Leukemic reticuloendotheliosis. Blood 13:609, 1958
2. Golomb Hh4, Hadad W: Infectious complications in 127 patients with hairy cell leukemia. Am J Hemato1 16:393, 1984
3. Juliusson G, Liliemark J: Rapid recovery from cytopenia in
hairy cell leukemia following treatment with 2-chloro-2’-deoxyadennosine (CdA): Relation to opportunistic infections. Blood 79:888,
1992
4. Melo JV, San Miguel JF, Moss VE, Catovsky D: The membrane phenotype of hairy cell leukemia: A study with monoclonal
antibodies. Semin Oncol 11:381, 1984
5. Juliusson G, Lenkei R, Liliemark J: Flow cytometry of blood
and bone marrow cells from patients with hairy cell leukemia. Phenotype of hairy cells and lymphocyte subsets following treatment
with 2-chlorodeoxyadenosine. Blood (in press)
6. Golomb HM, Lindgren V, Rowley JD: Chromosome abnormalities in patients with hairy cell leukemia. Cancer 41: 1374, 1978
7. Golomb HM, Lindgren V, Rowley JD:Hairy cell leukemia:
An analysis of the chromosomes of 26 patients. Virchows Arch B
Cell Path01 29:113, 1978
8. Ueshima Y,Alimena G, Rowley JD, Golomb I“: Cytogenetic
studies in patients with hairy cell leukemia. Hematol Oncol 1:215,
1983
9. Berger R, Bernheim A, Valensi F, Flandrin G: 14q- in two
hairy cell leukemia patients. Cancer Genet Cytogenet 16:91, 1985
10. Brito-Babapulle V, Pittman S , Melo JV, Parreira L, Catovsky
D: The 14q+ marker in hairy cell leukemia. A cytogenetic study of
15 cases. Leuk Res 10:131, 1986
11. Juliusson G, Gahrton G: Cytogenetics in CLL and related
disorders, in Rozman C (ed): Chronic Lymphocytic Leukaemia and
Related Disorders, Baillitre’s Clinical Haematology, v01 6. Philadelphia, PA, Saunders, 1993, p 821
12. Visser L, Shaw A, Slupsky J, Vos H, Poppema SI Monoclonal
antibodies reactive with hairy cell leukemia. Blood 74:320, 1989
13. Beutler E: Cladribine (2-chlorodeoxyadenosine). Lancet
340:952, 1992
14. Rosen A, Uggla C, Szigeti R, Kallin B, Lindqvist C, Zeuthen
J: A T-helper cell X Molt4 human hybridoma constitutively producing B-cell stimulatory and inhibitory factors. Lymph Res 5:185, 1986
15. Juliusson G, RobbrtK-H, Ost A, Friberg K, Biberfeld P,
Nilsson B, Zech L, Gahrton G: Prognostic information from cytogenetic analysis in chronic B-lymphocytic leukemia and leukemic immunocytoma. Blood 65:134, 1985
16. Tomiyasi T, Testa JR: Application of ethidium bromide for
high-resolution banding analysis of chromosomes from human malignant cells. Stain Techno1 63:83, 1988
17. Caspersson T, Lomakka G, Zech L The 24 fluorescence patterns of the human metaphase chromosomes-distinguishing characters and variability. Hereditas 67:89, 1971
18. Zhang S , Qiu J, Gao X: G-banding of human chromosomes.
Chin Med J 591210, 1979
19. Harnden DG, Klinger HP, (ed): ISCN 1985: An International
System for Human Cytogenetic Nomenclature. Basel, Switzerland,
Karger Basel, 1985
20. Mitelman F, (ed): ISCN 1991: Guidelines for Cancer Cytogenetics. Supplement to an International System for Human Cytogenetic Nomenclature. Basel, Switzerland, Karger Basel. 1991
21. Lewis JP, Tanke HJ, Raap AK, Kibbelaar RE, KluinPM,
Kluin-Nelemans HC: Hcell leukemia: An interphase cytogenetic
study. Leukemia 7:1334, 1993
22. Nishida K, Taniwaki M, Misawa S , Abe T: Nonrandom rearrangement of chromosome 14 at band q32.33 in human lymphoid
malignancies with mature B-cell phenotype. Cancer Res 49:1275,
1989
23. Nacheva E, Fischer P, O’Connor S , Bloxham D, Hoggarth
C, Marcus R: Complex chromosomal rearrangement in an unusual
variant of hairy cell leukemia. Cancer Genet Cytogenet 62: 186. 1992
24. Juliusson G, Gahrton G for the International Working Party
on Chromosomes in Chronic Lymphocytic Leukemia (WCCLL):
Chromosome abnormalities in B-cell chronic lymphocytic leukemia,
in(ed): Cheson BD Chronic Lymphocytic Leukemia, ch 4. New
York, NY, Marcel Dekker, 1992, p 83
25. Jacobs A: Genetic abnormalities in myelodysplastic syndrome. Cancer Genet Cytogenet 56:1, 1991
26. Le Beau MM: Cytogenetic and molecular analysis of the
deletions of chromosome 5 in myeloid disorders, in The Biology of
Hematopoiesis. New York, NY, Wiley-Liss. 1990, p 277
27. Gahrton G, Rob& K-H, Friberg K, Zech L, Bird AG: Nonrandom chromosomal aberrations in chronic lymphocytic leukemia revealedby polyclonal B-cell-mitogen stimulation. Blood 56640,
1980
28. Sadamori A. Sandberg AA: 14q+ and 6q- anomalies In a
case with hairy cell leukemia. Cancer Genet Cytogenet 8:89, L983
29. Mitelman F, Kaneko Y,Trent JM: Report of the committee
on chromosome changes in neoplasia. Human Gene Mapping 10.5.
Cytogenet Cell Genet 55:358, 1990
30. McKeithan TW, Rowley JD, Shows TB, Diaz MO: Cloning
of the chromosome translocation breakpoint junction of the t(14; 19)
in chronic lymphocytic leukemia. Proc Natl Acad Sci USA 84:9257,
1987
31. Bird ML, Ueshma Y, Rowley JD, Haren JH, Vardiman JW:
Chromosome abnormalities in B cell chronic lymphocytic leukemia
and their clinical correlations. Leukemia 3:182, 1989
32. Mecucci C, Rege-Cambrin G, Michaux J-L, Tricot G, van
den Berghe H: Multiple chromosomally distinct cell populations in
myelodysplastic syndromes and their possible significance in the
evolution of the disease. Br J Haematol 64:699, 1986
33. Heim S , Mitelman F: Cytogenetically unrelated clones in hematological neoplasms. Leukemia 316, 1989
34. Furuya T, Morgan R, Sandberg AA: Cytogenetic biclonality
in malignant disorders. Cancer Genet Cytogenet 62:25, 1992
35. Peterson LC, Lindquist LL, Church S , KayNE: Frequent
clonal abnormalities of chromosome band 13q14 in B-cell chronic
lymphocytic leukemia: Multiple clones, subclones, and nonclonal
alterations in 82 Midwestern patients. Genes Chrom Cancer 4:273,
1992
36. Whang-Peng J, Lee EC, Sieverts H, Magrath IT: Burlutt’s
lymphoma in AIDS: Cytogenetic study. Blood 63:818, 1984
37. Shippey CA, Layton M, Secker-Walker LM: Leukemia characterised by multiple subclones with unbalanced translocations involving different telomeric segments: Case report and review of the
literature. Genes Chrom Cancer 2:14, 1990
38. Ben-Neriah S , Abramov A, Lerer I, Polliack A, Leizerovilz
R, Rabinowitz R, Abeliovich D: “Jumping translocation” in a 17month-old child with mixed-lineage leukemia. Cancer Genet Cytogenet 56:223, 1991
39. Savage JRK: Application of chromosome banding techniques
to the study of primary chromosome structural changes. J Med Genet
14:362, 1977
40. Dix D: The role of ageing in cancer incidence: An epidemiological study. J Gerontology: 44:10, 1989
41. Fearon ER, Vogelstein B: A genetic model for colorectal
tumorigenesis. Cell 61:759, 1990
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
CHROMOSOMEABNORMALITIES IN HCL
42. Jacobs PA, Melville M, Ratcliffe S, KeayAJ, Syme J: A
cytogenetic survey of 11680 newborn infants. Ann Hum Genet
37:359, 1974
43. UNSCEAR Report: Ionizing radiation: sources and biological
effects. United Nations Scientific Committee on the Effects of
Atomic Radiation. New York, NY, United Nations, 1982
44. Benitez J, Valcarcel E, Ramos C, Ayuso C, Cascos AS: Frequency of constitutional chromosome alterations in patients with
hematologic neoplasias. Cancer Genet Cytogenet 24:345, 1987
45. Hsieh C-L, Vogel US, Dixon RAF, Francke U: Chromosome
localization and cDNA sequence of murine and human genes for
ras p21 GTPase activating protein (GAP). Somat Cell Mol Genet
15579, 1989
46. Wasmuth JJ, Bishop DT, Westbrook CA: Report of the committee on the genetic constitution of chromosome 5. Human Gene
Mapping 11. Cytogenet Cell Genet 58:261, 1991
2645
47. Spandidos DA, Freshney M, Wilkie NM: Heterogeneity of
cell lines derived after transformation of early passage rodent cells
by the Ha-rasl human oncogene. Anticancer Res 5:387, 1985
48. Bos JL: The ras gene family and human carcinogenesis. Mutat
Res 195:255, 1988
49. Barbacid M: ras genes. Ann Rev Biochem 56:779, 1987
50. Bmns GA, Dracopoli NC: Report of the committee on the
genetic constitution of chromosome 1. Human Gene Mapping 11.
Cytogenet Cell Genet 58:103, 1991
5 1. Kunz BA: Genetic effects of deoxyribonucleotide pool imbalances. Environ Mutagen 4695, 1982
52. Sutherland GR: Heritable fragile sites on human chromosomes I. Factors affecting expression in lymphocyte culture. Am J
Hum Genet 31:125, 1979
53. Hecht F, Ramesh K, Lockwood DH: A guide to fragile sites
on human chromosomes. Cancer Genet Cytogenet 44:37, 1990
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1994 83: 2637-2645
Hairy cell leukemia is characterized by clonal chromosome
abnormalities clustered to specific regions
U Haglund, G Juliusson, B Stellan and G Gahrton
Updated information and services can be found at:
http://www.bloodjournal.org/content/83/9/2637.full.html
Articles on similar topics can be found in the following Blood collections
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American
Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.